Heterocyclic sulfonamides as modulators of cardiac sarcomeres

ABSTRACT

Certain substituted sulfonamide derivatives of Formula (I) selectively modulate the cardiac sarcomere, for example by potentiating cardiac myosin, and are useful in the treatment of systolic heart failure including congestive heart failure.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of application Ser. No.10/550,398 with a 371 (c) date of Sep. 20, 2006 now U.S. Pat. No.7,595,322, which is the National Stage of International Application No.PCT/US04/09408, filed Mar. 26, 2004, and claims the benefit ofProvisional Application No. 60/458,702, filed Mar. 27, 2003; all ofwhich are incorporated herein by reference.

FIELD OF THE INVENTION

The invention relates to substituted sulfonamide derivatives,particularly to compounds that selectively modulate the cardiacsarcomere, and specifically to compounds, pharmaceutical formulationsand methods of treatment for systolic heart failure, includingcongestive heart failure.

BACKGROUND OF THE INVENTION

The Cardiac Sarcomere

The “sarcomere” is an elegantly organized cellular structure found incardiac and skeletal muscle made up of interdigitating thin and thickfilaments; it comprises nearly 60% of cardiac cell volume. The thickfilaments are composed of “myosin,” the protein responsible fortransducing chemical energy (ATP hydrolysis) into force and directedmovement. Myosin and its functionally related cousins are called motorproteins. The thin filaments are composed of a complex of proteins. Oneof these proteins, “actin” (a filamentous polymer) is the substrate uponwhich myosin pulls during force generation. Bound to actin are a set ofregulatory proteins, the “troponin complex” and “tropomyosin,” whichmake the actin-myosin interaction dependent on changes in intracellularCa²⁺ levels. With each heartbeat, Ca²⁺ levels rise and fall, initiatingcardiac muscle contraction and then cardiac muscle relaxation (Robbins Jand Leinwand L A. (1999) Molecular Basis of Cardiovascular Disease,Chapter 8. editor Chien, K. R., W. B. Saunders, Philadelphia). Each ofthe components of the sarcomere contributes to its contractile response.

Myosin is the most extensively studied of all the motor proteins. Of thethirteen distinct classes of myosin in human cells, the myosin-II classis responsible for contraction of skeletal, cardiac, and smooth muscle.This class of myosin is significantly different in amino acidcomposition and in overall structure from myosin in the other twelvedistinct classes (Goodson H V and Spudich J A. (1993) Proc. Natl. Acad.Sci. USA 90:659-663). Myosin-II consists of two globular head domainslinked together by a long alpha-helical coiled-coiled tail thatassembles with other myosin-IIs to form the core of the sarcomere'sthick filament. The globular heads have a catalytic domain where theactin binding and ATP functions of myosin take place. Once bound to anactin filament, the release of phosphate (cf. ATP to ADP) leads to achange in structural conformation of the catalytic domain that in turnalters the orientation of the light-chain binding lever arm domain thatextends from the globular head; this movement is termed the powerstroke.This change in orientation of the myosin head in relationship to actincauses the thick filament of which it is a part to move with respect tothe thin actin filament to which it is bound (Spudich J A. (2001) NatRev Mol Cell Biol. 2(5):387-92). Un-binding of the globular head fromthe actin filament (also Ca²⁺ modulated) coupled with return of thecatalytic domain and light chain to their startingconformation/orientation completes the contraction and relaxation cycle.

Mammalian heart muscle consists of two forms of cardiac myosin, alphaand beta, and they are well characterized (Robbins, supra). The betaform is the predominant form (>90 percent) in adult human cardiacmuscle. Both have been observed to be regulated in human heart failureconditions at both transcriptional and translational levels (Miyatasupra), with the alpha form being down-regulated in heart failure.

The sequences of all of the human skeletal, cardiac, and smooth musclemyosins have been determined. While the cardiac alpha and beta myosinsare very similar (93% identity), they are both considerably differentfrom human smooth muscle (42% identity) and more closely related toskeletal myosins (80% identity). Conveniently, cardiac muscle myosinsare incredibly conserved across mammalian species. For example, bothalpha and beta cardiac myosins are >96% conserved between humans andrats, and the available 250-residue sequence of porcine cardiac betamyosin is 100% conserved with the corresponding human cardiac betamyosin sequence. Such sequence conservation contributes to thepredictability of studying myosin based therapeutics in animal basedmodels of heart failure.

The components of the cardiac sarcomere present targets for thetreatment of heart failure, for example by increasing contractility orfacilitating complete relaxation to modulate systolic and diastolicfunction, respectively.

Heart Failure

Congestive heart failure (“CHF”) is not a specific disease, but rather aconstellation of signs and symptoms, all of which are caused by aninability of the heart to adequately respond to exertion by increasingcardiac output. The dominant pathophysiology associated with CHF issystolic dysfunction, an impairment of cardiac contractility (with aconsequent reduction in the amount of blood ejected with eachheartbeat). Systolic dysfunction with compensatory dilation of theventricular cavities results in the most common form of heart failure,“dilated cardiomyopathy,” which is often considered to be one in thesame as CHF. The counterpoint to systolic dysfunction is diastolicdysfunction, an impairment of the ability to fill the ventricles withblood, which can also result in heart failure even with preserved leftventricular function. Congestive heart failure is ultimately associatedwith improper function of the cardiac myocyte itself, involving adecrease in its ability to contract and relax.

Many of the same underlying conditions can give rise to systolic and/ordiastolic dysfunction, such as atherosclerosis, hypertension, viralinfection, valvular dysfunction, and genetic disorders. Patients withthese conditions typically present with the same classical symptoms:shortness of breath, edema and overwhelming fatigue. In approximatelyhalf of the patients with dilated cardiomyopathy, the cause of theirheart dysfunction is ischemic heart disease due to coronaryatherosclerosis. These patients have had either a single myocardialinfarction or multiple myocardial infarctions; here, the consequentscarring and remodeling results in the development of a dilated andhypocontractile heart. At times the causative agent cannot beidentified, so the disease is referred to as “idiopathic dilatedcardiomyopathy.” Irrespective of ischemic or other origin, patients withdilated cardiomyopathy share an abysmal prognosis, excessive morbidityand high mortality.

The prevalence of CHF has grown to epidemic proportions as thepopulation ages and as cardiologists have become more successful atreducing mortality from ischemic heart disease, the most common preludeto CHF. Roughly 4.6 million people in the United States have beendiagnosed with CHF; the incidence of such diagnosis is approaching 10per 1000 after 65 years of age. Hospitalization for CHF is usually theresult of inadequate outpatient therapy. Hospital discharges for CHFrose from 377,000 (in 1979) to 957,000 (in 1997) making CHF the mostcommon discharge diagnosis in people age 65 and over The five-yearmortality from CHF approaches 50% (Levy D. (2002) New Engl J Med.347(18):1442-4). Hence, while therapies for heart disease have greatlyimproved and life expectancies have extended over the last severalyears, new and better therapies continue to be sought, particularly forCHF.

“Acute” congestive heart failure (also known as acute “decompensated”heart failure) involves a precipitous drop in heart function resultingfrom a variety of causes. For example in a patient who already hascongestive heart failure, a new myocardial infarction, discontinuationof medications, and dietary indiscretions may all lead to accumulationof edema fluid and metabolic insufficiency even in the resting state. Atherapeutic agent that increases heart function during such an acuteepisode could assist in relieving this metabolic insufficiency andspeeding the removal of edema, facilitating the return to the morestable “compensated” congestive heart failure state. Patients with veryadvanced congestive heart failure particularly those at the end stage ofthe disease also could benefit from a therapeutic agent that increasesheart function, for example, for stabilization while waiting for a hearttransplant. Other potential benefits could be provided to patientscoming off a bypass pump, for example, by administration of an agentthat assists the stopped or slowed heart in resuming normal function.Patients who have diastolic dysfunction (insufficient relaxation of theheart muscle) could benefit from a therapeutic agent that modulatesrelaxation.

Therapeutic Active Agents

Inotropes are drugs that increase the contractile ability of the heart.As a group, all current inotropes have failed to meet the gold standardfor heart failure therapy, i.e., to prolong patient survival. Inaddition, current agents are poorly selective for cardiac tissue, inpart leading to recognized adverse effects that limit their use. Despitethis fact, intravenous inotropes continue to be widely used in acuteheart failure (e.g., to allow for reinstitution of oral medications orto bridge patients to heart transplantation) whereas in chronic heartfailure, orally given digoxin is used as an inotrope to relieve patientsymptoms, improve the quality of life, and reduce hospital admissions.

Given the limitations of current agents, new approaches are needed toimprove cardiac function in congestive heart failure. The most recentlyapproved short-term intravenous agent, milrinone, is now nearly fifteenyears old. The only available oral drug, digoxin, is over 200 hundredyears old. There remains a great need for agents that exploit newmechanisms of action and may have better outcomes in terms of relief ofsymptoms, safety, and patient mortality, both short-term and long-term.New agents with an improved therapeutic index over current agents willprovide a means to achieve these clinical outcomes.

The selectivity of agents directed at the cardiac sarcomere (forexample, by targeting cardiac beta myosin) has been identified as animportant means to achieve this improved therapeutic index. The presentinvention provides such agents (particularly sarcomere activatingagents) and methods for their identification and use.

SUMMARY OF THE INVENTION

The present invention provides compounds, pharmaceutical compositionsand methods for the treatment of heart failure including CHF,particularly systolic heart failure. The compositions are selectivemodulators of the cardiac sarcomere, for example, potentiating cardiacmyosin.

In one aspect, the invention relates to one or more compounds of thegroup represented by Formula I:

wherein:

-   -   R¹ and R² are independently selected from the group consisting        of hydrogen, optionally substituted alkyl, optionally        substituted aryl, optionally substituted heteroaryl, optionally        substituted aralkyl, and optionally substituted heteroaralkyl;        or R¹, R² and the nitrogen to which they are attached form an        optionally substituted 5-, 6-, or 7-membered heterocyclic ring;    -   R³ is an optionally substituted aryl or optionally substituted        heteroaryl;    -   R⁴ is halogen;    -   R⁵ is hydrogen, halogen, hydroxy, or optionally substituted        lower alkyl; and    -   R⁶ and R⁷ are independently selected from the group consisting        of hydrogen, halogen, hydroxy, and optionally substituted lower        alkyl;        including single stereoisomers, mixtures of stereoisomers, and        the pharmaceutically acceptable salts, solvates, and solvates of        pharmaceutically acceptable salts thereof. The compounds of        Formula I are useful as active agents in practice of the methods        of treatment and in manufacture of the pharmaceutical        formulations of the invention, and as intermediates in the        synthesis of such active agents.

Yet other aspects of the invention relate to a pharmaceuticalformulation including a pharmaceutically acceptable excipient, and to amethod of treatment for heart disease, each entailing a therapeuticallyeffective amount of a compound, isomer, salt or solvate represented byFormula I.

In an additional aspect, the present invention provides methods ofscreening for compounds that will bind to myosin (particularly myosin iior β myosin), for example compounds that will displace or compete withthe binding of the compounds of Formula I. The methods comprisecombining an optionally-labeled compound of Formula I, myosin, and atleast one candidate agent and determining the binding of the candidateagent to myosin.

In a further aspect, the invention provides methods of screening formodulators of the activity of myosin. The methods comprise combining acompound of Formula I, myosin, and at least one candidate agent anddetermining the effect of the candidate agent on the activity of myosin.

Other aspects and embodiments will be apparent to those skilled in theart form the following detailed description.

DETAILED DESCRIPTION

The present invention provides compounds useful in selective modulationof the cardiac sarcomere, for example, by potentiating cardiac myosin.The compounds can be used to treat heart failure including CHF,particularly systolic heart failure. The invention further relates topharmaceutical formulations comprising compounds of the invention, andto methods of treatment employing such compounds or compositions. Thecompositions are selective modulators of the cardiac sarcomere, forexample, potentiating cardiac myosin.

Definitions

As used in the present specification, the following words and phrasesare generally intended to have the meanings as set forth below, exceptto the extent that the context in which they are used indicatesotherwise. The following abbreviations and terms have the indicatedmeanings throughout:

-   -   Ac=acetyl    -   Boc=t-butyloxy carbonyl    -   c-=cyclo    -   CBZ=carbobenzoxy=benzyloxycarbonyl    -   DCM=dichloromethane=methylene chloride=CH₂Cl₂    -   DIEA=DIPEA=N,N-diisopropylethylamine    -   DMF=N,N-dimethylformamide    -   DMSO=dimethyl sulfoxide    -   Et=ethyl    -   EtOAc=ethyl acetate    -   EtOH=ethanol    -   GC=gas chromatograghy    -   h=hour    -   HATU=O-(7-azabenzotriazol-1-yl)-N,N,N′,N″-tetramethyluronium        hexafluorophosphate    -   HBTU=2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium        hexafluorophosphate    -   HOAt=7-aza-1-hydroxybenzotriazole    -   HOBt=1-Hydroxybenzotriazole    -   Me=methyl    -   min=minute    -   mL=milliliter    -   Ph=phenyl    -   rt=room temperature    -   s-=secondary    -   t-=tertiary    -   TES=triethylsilane    -   TFA=trifluoroacetic acid    -   THF=tetrahydrofuran    -   TLC=thin layer chromatography

The term “optional” or “optionally” means that the subsequentlydescribed event or circumstance may or may not occur, and that thedescription includes instances where said event or circumstance occursand instances in which it does not. For example, “optionally substitutedalkyl” means either “alkyl” or “substituted alkyl,” as defined below. Itwill be understood by those skilled in the art with respect to any groupcontaining one or more substituents that such groups are not intended tointroduce any substitution or substitution patterns that are stericallyimpractical, synthetically non-feasible and/or inherently unstable.

“Alkyl” is intended to include linear, branched, or cyclic hydrocarbonstructures and combinations thereof. Lower alkyl refers to alkyl groupsof from 1 to 5 carbon atoms. Examples of lower alkyl groups includemethyl, ethyl, propyl, isopropyl, butyl, s-and t-butyl and the like.Preferred alkyl groups are those of C₂₀ or below. More preferred alkylgroups are those of C₁₃ or below. Still more preferred alkyl groups arethose of C₆ and below. Cycloalkyl is a subset of alkyl and includescyclic hydrocarbon groups of from 3 to 13 carbon atoms. Examples ofcycloalkyl groups include c-propyl, c-butyl, c-pentyl, norbornyl,adamantyl and the like. In this application, alkyl refers to alkanyl,alkenyl and alkynyl residues; it is intended to includecyclohexylmethyl, vinyl, allyl, isoprenyl and the like. Alkylene isanother subset of alkyl, referring to the same residues, as alkyl, buthaving two points of attachment. Examples of alkylene include ethylene(—CH₂CH₂—), propylene (—CH₂CH₂CH₂—), dimethylpropylene (—CH₂C(CH₃)₂CH₂—)and cyclohexylpropylene (—CH₂CH₂CH(C₆H₁₃)—). When an alkyl residuehaving a specific number of carbons is named, all geometric isomershaving that number of carbons are intended to be encompassed; thus, forexample, “butyl” is meant to include n-butyl, sec-butyl, isobutyl andt-butyl; “propyl” includes n-propyl and isopropyl.

The term “alkoxy” or “alkoxyl” refers to the group —O-alkyl, preferablyincluding from 1 to 8 carbon atoms of a straight, branched, cyclicconfiguration and combinations thereof attached to the parent structurethrough an oxygen. Examples include methoxy, ethoxy, propoxy,isopropoxy, cyclopropyloxy, cyclohexyloxy and the like. Lower-alkoxyrefers to groups containing one to four carbons.

The term “substituted alkoxy” refers to the group —O-(substitutedalkyl). One preferred substituted alkoxy group is “polyalkoxy” or—O-(optionally substituted alkylene)-(optionally substituted alkoxy),and includes groups such as —OCH₂CH₂OCH₃, and glycol ethers such aspolyethyleneglycol and —O(CH₂CH₂O)_(x)CH₃, where x is an integer ofabout 2-20, preferably about 2-10, and more preferably about 2-5.Another preferred substituted alkoxy group is hydroxyalkoxy or—OCH₂(CH₂)_(y)OH, where y is an integer of about 1-10, preferably about1-4.

“Acyl” refers to groups of from 1 to 10 carbon atoms of a straight,branched, cyclic configuration, saturated, unsaturated and aromatic andcombinations thereof, attached to the parent structure through acarbonyl functionality. One or more carbons in the acyl residue may bereplaced by nitrogen, oxygen or sulfur as long as the point ofattachment to the parent remains at the carbonyl. Examples includeacetyl, benzoyl, propionyl, isobutyryl, t-butoxycarbonyl,benzyloxycarbonyl and the like. “Lower-acyl” refers to groups containingone to four carbons and “acyloxy” refers to the group O-acyl.

The term “amino” refers to the group —NH₂. The term “substituted amino”refers to the group —NHR or —NRR where each R is independently selectedfrom the group: optionally substituted alkyl, optionally substitutedalkoxy, optionally substituted amino, optionally substituted aryl,optionally substituted heteroaryl, optionally substituted heterocyclyl,acyl, alkoxycarbonyl, sulfanyl, sulfinyl and sulfonyl, e.g.,diethylamino, methylsulfonylamino, furanyl-oxy-sulfonamino.

“Aryl” means a 5- or 6-membered aromatic ring, a bicyclic 9- or10-membered aromatic ring system, or a tricyclic 12- to 14-memberedaromatic ring system. Examples include cyclopenta-1,3-diene, phenyl,naphthyl, indane, tetraline, fluorene, cyclopenta[b]naphthalene andanthracene;

“Aralkoxy” refers to the group —O-aralkyl. Similarly, “heteroaralkoxy”refers to the group —O-heteroaralkyl; “aryloxy” refers to —O-aryl; and“heteroaryloxy” refers to the group —O-heteroaryl.

“Aralkyl” refers to a residue in which an aryl moiety is attached to theparent structure via an alkyl residue. Examples include benzyl,phenethyl, phenylvinyl, phenylallyl and the like. “Heteroaralkyl” refersto a residue in which a heteroaryl moiety is attached to the parentstructure via an alkyl residue. Examples include furanylmethyl,pyridinylmethyl, pyrimidinylethyl and the like.

“ATPase” refers to an enzyme that hydrolyzes ATP. ATPases includeproteins comprising molecular motors such as the myosins.

“Halogen” or “halo” refers to fluorine, chlorine, bromine or iodine.Fluorine, chlorine and bromine are preferred. Dihaloaryl, dihaloalkyl,trihaloaryl etc. refer to aryl and alkyl substituted with a plurality ofhalogens, but not necessarily a plurality of the same halogen; thus4-chloro-3-fluorophenyl is within the scope of dihaloaryl.

“Heteroaryl” means a 5- or 6-membered aromatic ring containing 1-4heteroatoms, a bicyclic 8-, 9- or 10-membered aromatic ring systemcontaining 1-4 (or more) heteroatoms, or a tricyclic 11- to 14-memberedaromatic ring system containing 1-4 (or more) heteroatoms; theheteroatoms are selected from O, N and S. Examples include furan,pyrrole, thiophene, pyrazole, imidazole, triazole, tetrazole, dithiole,oxazole, isoxazole, oxadiazole, thiazole, thiopyran, pyridine,pyridazine, pyrimidine, pyrazine, indole, benzofuran, benzothiophene,quinoline, isoquinoline and quinoxaline.

“Heterocycle” or “heterocyclyl” refers to a cycloalkyl residue in whichone to four of the carbons is replaced by a heteroatom such as oxygen,nitrogen or sulfur. a 4-, 5-, 6- or 7-membered non-aromatic ringcontaining 1-4 heteroatoms, a bicyclic 8-, 9- or 10-memberednon-aromatic ring system containing 1-4 (or more) heteroatoms, or atricyclic 11- to 14-membered non-aromatic ring system containing 1-4 (ormore) heteroatoms; the heteroatoms are selected from O, N and S.Examples include pyrrolidine, tetrahydrofuran, tetrahydro-thiophene,thiazolidine, piperidine, tetrahydro-pyran, tetrahydro-thiopyran,piperazine, morpholine, thiomorpholine and dioxane. Heterocyclyl alsoincludes ring systems including unsaturated bonds, provided the numberand placement of unsaturation does not render the group aromatic.Examples include imidazoline, oxazoline, tetrahydroisoquinoline,benzodioxan, benzodioxole and 3,5-dihydrobenzoxazinyl. Examples ofsubstituted heterocyclyl include 4-methyl-1-piperazinyl and 4-benzyl-1-piperidinyl.

“Isomers” are different compounds that have the same molecular formula.“Stereoisomers” are isomers that differ only in the way the atoms arearranged in space. “Enantiomers” are a pair of stereoisomers that arenon-superimposable mirror images of each other. A 1:1 mixture of a pairof enantiomers is a “racemic” mixture. The term “(.±.)” is used todesignate a racemic mixture where appropriate. “Diastereoisomers” arestereoisomers that have at least two asymmetric atoms, but which are notmirror-images of each other. The absolute stereochemistry is specifiedaccording to the Cahn-Ingold-Prelog R-S system. When a compound is apure enantiomer the stereochemistry at each chiral carbon may bespecified by either R or S. Resolved compounds whose absoluteconfiguration is unknown can be designated (+) or (−) depending on thedirection (dextro- or levorotatory) which they rotate plane polarizedlight at the wavelength of the sodium D line. Certain of the compoundsdescribed herein contain one or more asymmetric centers and may thusgive rise to enantiomers, diastereomers, and other stereoisomeric formsthat may be defined, in terms of absolute stereochemistry, as (R)- or(S)-. The present invention is meant to include all such possibleisomers, including racemic mixtures, optically pure forms andintermediate mixtures. Optically active (R)- and (S)-isomers may beprepared using chiral synthons or chiral reagents, or resolved usingconventional techniques. When the compounds described herein containolefinic double bonds or other centers of geometric asymmetry, andunless specified otherwise, it is intended that the compounds includeboth E and Z geometric isomers. Likewise, all tautomeric forms are alsointended to be included.

The term “pharmaceutically acceptable carrier” or “pharmaceuticallyacceptable excipient” includes any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents and the like. The use of such media and agents forpharmaceutically active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive ingredient, its use in the therapeutic compositions iscontemplated. Supplementary active ingredients can also be incorporatedinto the compositions.

The term “pharmaceutically acceptable salt” refers to salts that retainthe biological effectiveness and properties of the compounds of thisinvention and, which are not biologically or otherwise undesirable. Inmany cases, the compounds of this invention are capable of forming acidand/or base salts by virtue of the presence of amino and/or carboxylgroups or groups similar thereto. Pharmaceutically acceptable acidaddition salts can be formed with inorganic acids and organic acids.Inorganic acids from which salts can be derived include, for example,hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like. Organic acids from which salts can bederived include, for example, acetic acid, propionic acid, glycolicacid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinicacid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamicacid, mandelic acid, methanesulfonic acid, ethanesulfonic acid,p-toluenesulfonic acid, salicylic acid, and the like. Pharmaceuticallyacceptable base addition salts can be formed with inorganic and organicbases. Inorganic bases from which salts can be derived include, forexample, sodium, potassium, lithium, ammonium, calcium, magnesium, iron,zinc, copper, manganese, aluminum, and the like; particularly preferredare the ammonium, potassium, sodium, calcium and magnesium salts.Organic bases from which salts can be derived include, for example,primary, secondary, and tertiary amines, substituted amines includingnaturally occurring substituted amines, cyclic amines, basic ionexchange resins, and the like, specifically such as isopropylamine,trimethylamine, diethylamine, triethylamine, tripropylamine, andethanolamine.

The term “solvate” refers to a compound (e.g., a compound of Formula Ior a pharmaceutically acceptable salt thereof) in physical associationwith one or more molecules of a pharmaceutically acceptable solvent. Itwill be understood that phrases such as “a compound of Formula I or apharmaceutically acceptable salt or solvate thereof” are intended toencompass the compound of Formula I, a pharmaceutically acceptable saltof the compound, a solvate of the compound, and a solvate of apharmaceutically acceptable salt of the compound.

“Substituted-”alkyl, aryl, heteroaryl and heterocyclyl referrespectively to alkyl, aryl, heteroaryl and heterocyclyl wherein one ormore (up to about 5, preferably up to about 3) hydrogen atoms arereplaced by a substituent independently selected from the group: acyl,optionally substituted alkyl (e.g., fluoroalkyl), optionally substitutedalkoxy, alkylenedioxy (e.g. methylenedioxy), optionally substitutedamino (e.g., alkylamino and dialkylamino), optionally substitutedamidino, optionally substituted aryl (e.g., phenyl), optionallysubstituted aralkyl (e.g., benzyl), optionally substituted aryloxy(e.g., phenoxy), optionally substituted aralkoxy (e.g., benzyloxy),carboxy (—COOH), acyloxy (—OOCR), alkoxycarbonyl (i.e., esters or—COOR), aminocarbonyl, benzyloxycarbonylamino (CBZ-amino), cyano,carbonyl, halogen, hydroxy, optionally substituted heteroaryl,optionally substituted heteroaralkyl, optionally substitutedheteroaryloxy, optionally substituted heteroaralkoxy, nitro, sulfanyl,sulfinyl, sulfonyl, and thio.

The term “sulfanyl” refers to the groups: —S-(optionally substitutedalkyl), —S-(optionally substituted aryl), —S-(optionally substitutedheteroaryl), and —S-(optionally substituted heterocyclyl).

The term “sulfinyl” refers to the groups: —S(O)—H, —S(O)-(optionallysubstituted alkyl), —S(O)-(optionally substituted amino),—S(O)-(optionally substituted aryl), —S(O)-(optionally substitutedheteroaryl), and —S(O)-(optionally substituted heterocyclyl).

The term “sulfonyl” refers to the groups: —S(O₂)—H, —S(O₂)-(optionallysubstituted alkyl), —S(O₂)-(optionally substituted amino),—S(O₂)-(optionally substituted aryl), —S(O₂)-(optionally substitutedheteroaryl), —S(O₂)-(optionally substituted heterocyclyl),—S(O₂)-(optionally substituted alkoxy), —S(O₂)-optionally substitutedaryloxy), —S(O₂)-(optionally substituted heteroaryloxy), and—S(O₂)-(optionally substituted heterocyclyloxy).

The term “therapeutically effective amount” or “effective amount” refersto that amount of a compound of Formula I that is sufficient to effecttreatment, as defined below, when administered to a mammal in need ofsuch treatment. The therapeutically effective amount will vary dependingupon the subject and disease condition being treated, the weight and ageof the subject, the severity of the disease condition, the particularcompound of Formula I chosen, the dosing regimen to be followed, timingof administration, the manner of administration and the like, all ofwhich can readily be determined by one of ordinary skill in the art.

The term “treatment” or “treating” means any treatment of a disease in amammal, including:

-   -   a) preventing the disease, that is, causing the clinical        symptoms of the disease not to develop;    -   b) inhibiting the disease, that is, slowing or arresting the        development of clinical symptoms; and/or    -   c) relieving the disease, that is, causing the regression of        clinical symptoms

Compounds of the Present Invention

The present invention is directed to the compounds that are selectivemodulators of the cardiac sarcomere (e.g., by stimulating or otherwisepotentiating the activity of cardiac myosin), as represented by FormulaI:

wherein:

-   -   R¹ and R² are independently selected from the group consisting        of hydrogen, optionally substituted alkyl, optionally        substituted aryl, optionally substituted heteroaryl, optionally        substituted aralkyl, and optionally substituted heteroaralkyl;        or R¹, R² and the nitrogen to which they are attached form an        optionally substituted 5-, 6-, or 7-membered heterocyclic ring;    -   R³ is optionally substituted aryl or optionally substituted        heteroaryl;    -   R⁴ is halogen;    -   R⁵ is hydrogen, halogen, hydroxy, or optionally substituted        lower alkyl; and    -   R⁶ and R⁷ are independently selected from the group consisting        of hydrogen, halogen, hydroxy, and optionally substituted lower        alkyl;        including single stereoisomers, mixtures of stereoisomers, and        the pharmaceutically acceptable salts thereof. The compounds of        Formula I are useful as active agents in practice of the methods        of treatment and in manufacture of the pharmaceutical        formulations of the invention, and as intermediates in the        synthesis of such active agents.        Nomenclature

The compounds of Formula I can be named and numbered (e.g., usingAutoNom version 2.2) as described below. For example, the compound:

i.e., the compound according to Formula I where R¹ and R² together withthe nitrogen to which they are attached form a substituted piperazinering; R³ is a substituted thiadiazole ring; R⁴ is chloro; and R⁵, R⁶,and R⁷ are hydrogen can be named4-[4-chloro-3-(5-phenyl-[1,3,4]thiadiazol-2-ylcarbamoyl)-benzenesulfonyl]-piperazine-1-carboxylicacid ethyl ester.

Likewise, the compound:

i.e., the compound according to Formula I where R¹ and R² together withthe nitrogen to which they are attached form a substituted piperazinering; R³ is an imidazole ring; R⁴ is chloro; and R⁵, R⁶, and R⁷ arehydrogen can be named2-chloro-5-[4-(N-cyclopentyl-N′-cyano-carbamimidoyl)-piperazine-1-sulfonyl]-N-(1H-imidazol-2-yl)-benzamide.

Synthesis of the Compounds of Formula I

The compounds of the invention can be synthesized utilizing techniqueswell known in the art, e.g., as illustrated below with reference to theReaction Schemes.

Synthetic Reaction Parameters

Unless specified to the contrary, the reactions described herein takeplace at atmospheric pressure, generally within a temperature range from−10° C. to 110° C. Further, except as employed in the Examples or asotherwise specified, reaction times and conditions are intended to beapproximate, e.g., taking place at about atmospheric pressure within atemperature range of about −10° C. to about 110° C. over a period ofabout 1 to about 24 hours; reactions left to run overnight average aperiod of about 16 hours.

The terms “solvent”, “organic solvent” or “inert solvent” each mean asolvent inert under the conditions of the reaction being described inconjunction therewith [including, for example, benzene, toluene,acetonitrile, tetrahydrofuran (“THF”), dimethylformamide (“DMF”),chloroform, methylene chloride (or dichloromethane), diethyl ether,methanol, pyridine and the like]. Unless specified to the contrary, thesolvents used in the reactions of the present invention are inertorganic solvents.

Isolation and purification of the compounds and intermediates describedherein can be effected, if desired, by any suitable separation orpurification procedure such as, for example, filtration, extraction,crystallization, column chromatography, thin-layer chromatography orthick-layer chromatography, or a combination of these procedures.Specific illustrations of suitable separation and isolation procedurescan be had by reference to the examples hereinbelow. However, otherequivalent separation or isolation procedures can, of course, also beused.

When desired, the (R)- and (S)-isomers may be resolved by methods knownto those skilled in the art, for example by formation ofdiastereoisomeric salts or complexes which may be separated, forexample, by crystallization; via formation of diastereoisomericderivatives which may be separated; for example, by cyrstallization,gas-liquid or liquid chromatography; selective reaction of oneenantiomer with an enantiomer-specific reagent, for example enzymaticoxidation or reduction, followed by separation of the modified andunmodified enantiomers; or gas-liquid or liquid chromatography in achiral environment, for example on a chiral support, such as silica witha bound chiral ligand or in the presence of a chiral solvent. Forexample, a compound of Formula I can be dissolved in a lower alkanol andplaced on a Chiralpak AD (205×20 mm) column (Chiral Technologies, Inc.)conditioned for 60 min at 70% EtOAc in Hexane. It will be appreciatedthat where the desired enantiomer is converted into another chemicalentity by one of the separation procedures described above, a furtherstep may be required to liberate the desired enantiomeric form.Alternatively, a specific enantiomer may be synthesized by asymmetricsynthesis using optically active reagents, substrates, catalysts orsolvents, or by converting one enantiomer to the other by asymmetrictransformation.

Starting Materials

The optionally substituted benzoic acids of Formula 101 are commerciallyavailable, e.g., from Acros Organic or Aldrich Chemical Company,Milwaukee, Wis., or may be readily prepared by those skilled in the artusing commonly employed synthetic methodology. One of skill in the artwill appreciate that the commercially available compounds may lack acarboxyl protecting group PG. Other reactants are likewise commerciallyavailable or may be readily prepared by those skilled in the art usingcommonly employed synthetic methodology.

Preparation of Compounds of Formula 103

Referring to Reaction Scheme 1, Step 1, a compound of Formula 101 isplaced in a vial. The vial is flushed with nitrogen and a positivepressure is maintained. An anhydrous, nonpolar solvent, such asdichloromethane is added, followed by an excess (preferably about 1.2equivalents) of a compound of formula R¹R²NH and a base such asethyldiisopropylamine. The mixture is stirred for about 1.5 hours afterwhich time an additional aliquot (preferably about 0.8 equivalent) ofthe amine of formula R¹R²NH is added. After about 14 hours the mixtureis analyzed by reverse-phase HPLC-MS in negative ionization mode. If thesulfonyl fluoride starting material is present, an additional amount(preferably about 0.35 equivalent) of the amine of formula R¹R²NH andethyldiisopropylamine are added and the mixture is stirred for about 4hours. This may be repeated again as necessary. The resulting product, acompound of Formula 103, can be recovered by conventional methods, suchas chromatography, filtration, evaporation, crystallization, and thelike or, alternatively, used in the next step without purificationand/or isolation. It should be noted that addition of excessnucleophilic amine at the beginning of the reaction may result insignificant bis addition, giving sulfonamide and carboxamide product.Stepwise addition of nucleophilic amine as needed suppresses formationof this side product.

Preparation of Compounds of Formula 105

Referring to Reaction Scheme 1, Step 2, a compound of Formula 103 isplaced in a vial along with an excess (preferably about 1.2 equivalents)of a compound of Formula R³NH₂, an excess (preferably about 1.5equivalents) of HBTU and an excess (preferably about 1.5 equivalents) ofHOBt hydrate. The vial is flushed with nitrogen and a positive pressureis maintained. An anhydrous solvent, such as dimethylformamide is added,followed by a base such as ethyldiisopropylamine and the mixture isstirred for about 14 hours. The resulting product, a compound of Formula105, can be recovered by conventional methods, such as chromatography,filtration, evaporation, crystallization, and the like or,alternatively, used in the next step without purification and/orisolation.

Preparation of Compounds of Formula 203

Referring to Reaction Scheme 2, Step 1, an excess (preferably about 10equivalents) of diphenylcyanocarbonimidate is added to a compound ofFormula 201 wherein n is 1 or 2. The vial is capped, flushed withnitrogen and a positive pressure is maintained. Anhydrous, inert solventsuch as THF is added, followed by a base such as ethyldiisopropylamine.The mixture is stirred for about one hour. The resulting product, acompound of Formula 203, can be recovered by conventional methods, suchas chromatography, filtration, evaporation, crystallization, and thelike or, alternatively, used in the next step without purificationand/or isolation.

Preparation of Compounds of Formula 205

Referring to Reaction Scheme 2, Step 2, a compound of Formula 203 isplaced in a vial. The vial is flushed with nitrogen and a positivepressure is maintained. An anhydrous inert solvent such as THF is addedfollowed by an excess (especially about five equivalents) of an amine offormula R¹⁰NH₂ wherein R¹⁰ is optionally substituted alkyl, optionallysubstituted aryl, optionally substituted aralkyl, optionally substitutedheteroaryl, or optionally substituted heteroaralkyl. The mixture isstirred until the reaction is complete. The resulting product, acompound of Formula 205, can be recovered by conventional methods, suchas chromatography, filtration, evaporation, crystallization, and thelike or, alternatively, used in the next step without purificationand/or isolation.

Compounds prepared by the above-described, processes of the inventioncan be identified, e.g., by the presence of a detectable amount of oneor more of the starting materials or reagents. While it is well knownthat pharmaceuticals must meet pharmacopoeia standards before approvaland/or marketing, and that synthetic reagents (such as the varioussubstituted amines or alcohols) and precursors should not exceed thelimits prescribed by pharmacopoeia standards, final compounds preparedby a process of the present invention may have minor, but detectable,amounts of such materials present, for example at levels in the range of95% purity with no single impurity greater than 1%. These levels can bedetected, e.g., by emission spectroscopy. It is important to monitor thepurity of pharmaceutical compounds for the presence of such materials,which presence is additionally disclosed as a method of detecting use ofa synthetic process of the invention.

Particular Processes and Last Steps

A racemic mixture of isomers of a compound of Formula I is placed on achromatography column and separated into (R)- and (S)-enantiomers.

A compound of Formula I is contacted with a pharmaceutically acceptableacid to form the corresponding acid addition salt.

A pharmaceutically acceptable acid addition salt of Formula I iscontacted with a base to form the corresponding free base of Formula I.

Particular Compounds

Provided for the compounds, pharmaceutical formulations, methods ofmanufacture and use of the present invention are the followingcombinations and permutations of substituent groups of Formula I.Particular embodiments of the invention include or employ the compoundsof Formula I having the following combinations and permutations ofsubstituent groups. These are presented in support of the appendedclaims to support other combinations and permutations of substituentgroups, which for the sake of brevity have not been specificallyclaimed, but should be appreciated as encompassed within the teachingsof the present disclosure.

R¹ and R²

When referring to compounds of Formula I, in a particular embodiment, R¹and R² are independently selected from the group consisting of hydrogen,optionally substituted alkyl, optionally substituted aryl, optionallysubstituted heteroaryl, optionally substituted aralkyl, and optionallysubstituted heteroaralkyl. [Brad—any sub-preferences for the non-cyclicversion?]

When referring to compounds of Formula I, in another particularembodiment, R¹, R² and the nitrogen to which they are attached form anoptionally substituted 5-, 6-, or 7-membered heterocyclic ring. In amore particular embodiment, R¹, R² and the nitrogen to which they areattached form a piperidin-1-yl; piperazin-1-yl; morpholin-4-yl;pyrrolidin-1-yl; thiomorpholin-4-yl or diazepan-1-yl, which optionallyis substituted with one, two or three of the following groups:optionally substituted alkyl, halogen, hydroxy, alkoxy, alkylenedioxy(e.g. methylenedioxy), carboxy (—COOH), optionally substituted acyloxy(RCOO—), optionally substituted alkoxycarbonyl- (—COOR), optionallysubstituted aminocarbonyl, cyano, optionally substituted acyl, oxo,nitro, optionally substituted amino, sulfanyl, sulfinyl, sulfonyl,optionally substituted aminosulfonyl-, amidino, phenyl, benzyl,heteroaryl, heterocyclyl, substituted heterocyclyl, aryloxy, arallkoxy,heteroaryloxy, and heteroaralkoxy.

When R¹, R² and the nitrogen to which they are attached form anoptionally substituted diazepan-1-yl ring, in a particular embodiment,the diazepane nitrogen is further substituted with optionallysubstituted acyl, optionally substituted alkoxycarbonyl, or optionallysubstituted aminosulfonyl.

When R¹, R² and the nitrogen to which they are attached form anoptionally substituted piperazin-1-yl ring, in a particular embodiment,the piperazine nitrogen is further substituted with hydrogen, anoptionally substituted acyl, optionally substituted alkoxycarbonyl,optionally substituted aminosulfonyl, optionally substituted heteroaryl,optionally substituted alkyl, or optionally substituted sulfonyl.

When R¹, R² and the nitrogen to which they are attached form anoptionally substituted piperadin-1-yl ring, in a particular embodiment,the piperidine ring is further substituted with hydrogen, optionallysubstituted alkoxycarbonyl, optionally substituted aminocarbonyl,optionally substituted amino, hydroxy, optionally substituted alkoxy, oralkylenedioxy.

When R¹, R² and the nitrogen to which they are attached form anoptionally substituted pyrrolidin-1-yl ring, in a particular embodiment,the pyrrolidine ring is further substituted with optionally substitutedamino.

R³

When referring to compounds of Formula I, in a particular embodiment, R³is optionally substituted aryl or optionally substituted heteroaryl.More particularly, R³ is phenyl, isoxazolyl, oxazolyl, pyridinyl,pyrazinyl, pyrimidinyl, tetrazol-5-yl, thiazolyl, thiadiazolyl orimidazolyl, which is optionally substituted with a halogen, loweralkoxy, an optionally substituted aryl or heteroaryl group.

In one particular embodiment, R³ is phenyl which is optionallysubstituted with halogen (especially fluoro) or lower alkoxy (especiallymethoxy). In another particular embodiment, R³ is a heteroaryl groupwhich is optionally substituted with an optionally substituted aryl orheteroaryl group. Yet more particularly, R³ is [1,3,4]thiadiazol-2-ylwhich is optionally substituted with an optionally substituted phenylgroup or R³ is 1H-imidazol-2-yl. In a most particular embodiment, R³ isoxazol-2-yl, 5-phenyl-[1,3,4]thiadiazol-2-yl or 1H-imidazol-2-yl.

R⁴

When referring to compounds of Formula I, in a particular embodiment, R⁴is halogen. More particularly, R⁴ is chloro.

R⁵, R⁶, and R⁷

When referring to compounds of Formula I, in a particular embodiment, R⁵is hydrogen, halogen, hydroxy, or optionally substituted lower alkyl;and R⁶ and R⁷ are independently selected from the group consisting ofhydrogen, hydrogen, halogen, hydroxy, and optionally substituted loweralkyl. In another embodiment, R⁵, R⁶ and R⁷ are hydrogen.

Particular Subgenus

When considering the compounds of Formula I, in a particular embodiment,

R¹, R² and the nitrogen to which they are attached form an optionallysubstituted 5-, 6-, or 7-membered heterocyclic ring;

R³ is optionally substituted aryl or optionally substituted heteroaryl;

R⁴ is halogen; and

R⁵, R⁸ and R⁷ are hydrogen.

In another particular embodiment,

R¹, R² and the nitrogen to which they are attached form an optionallysubstituted 5-, 6-, or 7-membered heterocyclic ring;

R³ is optionally substituted aryl or optionally substituted heteroaryl;

R⁴ is chloro; and

R⁵, R⁶ and R⁷ are hydrogen.

In another particular embodiment,

R¹, R² and the nitrogen to which they are attached form an optionallysubstituted 5-, 6-, or 7-membered heterocyclic ring;

R³ is [1,3,4]thiadiazol-2-yl which is optionally substituted with anoptionally substituted phenyl group or R³ is 1H-imidazol-2-yl group;

R⁴ is a halogen; and

R⁵, R⁶ and R⁷ are hydrogen.

In another particular embodiment,

R¹, R² and the nitrogen to which they are attached form an optionallysubstituted 5-, 6-, or 7-membered heterocyclic ring;

R³ is [1,3,4]thiadiazol-2-yl which is optionally substituted with anoptionally substituted phenyl group or R³ is a 1H-imidazol-2-yl group;

R⁴ is chloro; and

R⁵, R⁸ and R⁷ are hydrogen.

In another particular embodiment,

R¹, R² and the nitrogen to which they are attached form an optionallysubstituted 5-, 6-, or 7-membered heterocyclic ring;

R³ is 5-phenyl-[1,3,4]thiadiazol-2-yl or 1H-imidazol-2-yl;

R⁴ is halogen; and

R⁵, R⁶ and R⁷ are hydrogen.

In another particular embodiment,

R¹, R² and the nitrogen to which they are attached form an optionallysubstituted 5-, 6-, or 7-membered heterocyclic ring;

R³ is 5-phenyl-[1,3,4]thiadiazol-2-yl or 1H-imidazol-2-yl;

R⁴ is chloro; and

R⁵, R⁶ and R⁷ are hydrogen.

Particular compounds include

X R⁸ R⁹ R³ R⁴ R⁵ R⁶ R⁷ CHR⁹NR⁸ t-Butoxycarbonyl- H 1H-imidazol-2-yl Cl HH H CHR⁹NR⁸ Acetyl- H 1H-imidazol-2-yl Cl H H H CHR⁹NR⁸i-Propoxycarbonyl- H 1H-imidazol-2-yl Cl H H H CHR⁹NR⁸ Methoxycarbonyl-H 1H-imidazol-2-yl Cl H H H CHR⁹NR⁸ 3-Methylbutanoyl- H 1H-imidazol-2-ylCl H H H CHR⁹NR⁸ Isobutyryl- H 1H-imidazol-2-yl Cl H H H CHR⁹NR⁸Cyclopropylacetyl- H 1H-imidazol-2-yl Cl H H H CHR⁹NR⁸Dimethylaminosulfonyl- H 1H-imidazol-2-yl Cl H H H CHR⁹NR⁸sec-Butoxy-carbonyl- H 1H-imidazol-2-yl Cl H H H CHR⁹NR⁸ Propanoyl- H1H-imidazol-2-yl Cl H H H CHR⁹NR⁸ Cyclohexyloxycarbonyl- H1H-imidazol-2-yl Cl H H H CHR⁹NR⁸ Acetyl- H 5-Phenyl- Cl H H H[1,3,4]thiadiazol- 2-yl CR⁹R⁸ Hydrogen H 5-Phenyl- Cl H H H[1,3,4]thiadiazol- 2-yl CR⁹R⁸ Hydrogen H 5-Phenyl- Cl H H H[1,3,4]thiadiazol- 2-yl CR⁹R⁸ Methoxycarbonyl- H 5-Phenyl- Cl H H H[1,3,4]thiadiazol- 2-yl CR⁹R⁸ Hydrogen H 1H-imidazol-2-yl Cl H H H CR⁹R⁸Carbamoyl- H 1H-imidazol-2-yl Cl H H H CR⁹R⁸ Methoxycarbonyl- H1H-imidazol-2-yl Cl H H H CR⁹R⁸ N-(t-butoxycarbonyl)-N- H1H-imidazol-2-yl Cl H H H methylamino- CR⁹R⁸ Acetamido- H1H-imidazol-2-yl Cl H H H CR⁹R⁸ N-acetyl-N-methylamino- H1H-imidazol-2-yl Cl H H H CR⁹R⁸ Methylaminocarbonyl- H 1H-imidazol-2-ylCl H H H CR⁹R⁸ Ethoxycarbonyl- H 1H-imidazol-2-yl Cl H H H CR⁹R⁸ OH H1H-imidazol-2-yl Cl H H H CR⁹R⁸ N-(t-butoxycarbonyl)amino- H 5-Phenyl-Cl H H H [1,3,4]thiadiazol- 2-yl CR⁹R⁸ Methoxycarbonyl- H 5-Phenyl- Cl HH H [1,3,4]thiadiazol- 2-yl CR⁹R⁸ Carbamoyl- H 5-Phenyl- Cl H H H[1,3,4]thiadiazol- 2-yl CR⁹R⁸ Hydroxy- H 5-Phenyl- Cl H H H[1,3,4]thiadiazol- 2-yl CR⁹R⁸ Ethylenedioxy- 5-Phenyl- Cl H H H[1,3,4]thiadiazol- 2-yl CR⁹R⁸ Ethylenedioxy- 1H-imidazol-2-yl Cl H H HCR⁹R⁸ Ethylenedioxy- 5-Phenyl- Cl H H H [1,3,4]thiadiazol- 2-yl NR⁸Ethoxycarbonyl- — 5-Phenyl- Cl H H H [1,3,4]thiadiazol- 2-yl NR⁸ Acetyl-— 5-Phenyl- Cl H H H [1,3,4]thiadiazol- 2-yl NR⁸ Methoxycarbonyl- —5-Phenyl- Cl H H H [1,3,4]thiadiazol- 2-yl NR⁸ Methoxyacetyl- —5-Phenyl- Cl H H H [1,3,4]thiadiazol- 2-yl NR⁸ 3-Methylbutanoyl- —5-Phenyl- Cl H H H [1,3,4]thiadiazol- 2-yl NR⁸ Propoxycarbonyl- —5-Phenyl- Cl H H H [1,3,4]thiadiazol- 2-yl NR⁸ i-Propoxycarbonyl- —5-Phenyl- Cl H H H [1,3,4]thiadiazol- 2-yl NR⁸ Dimethylaminocarbonyl- —5-Phenyl- Cl H H H [1,3,4]thiadiazol- 2-yl NR⁸ Propoxycarbonyl- —1H-imidazol-2yl Cl H H H NR⁸ sec-Butoxycarbonyl- — 1H-imidazol-2yl Cl HH H (especially the R-isomer) NR⁸ 2-Cyclopentylacetyl- — 1H-imidazol-2ylCl H H H NR⁸ 2-Cyclohexylacetyl- — 1H-imidazol-2yl Cl H H H NR⁸Ethoxycarbonyl- — 5-Phenyl- Cl H H H [1,3,4]thiadiazol- 2-yl NR⁸Methoxyacetyl- — 5-Phenyl- Cl H H H [1,3,4]thiadiazol- 2-yl NR⁸ Acetyl-— 5-Phenyl- Cl H H H [1,3,4]thiadiazol- 2-yl NR⁸ Methyl- — 5-Phenyl- ClH H H [1,3,4]thiadiazol- 2-yl NR⁸ Ethoxycarbonyl- — Fluorophenyl- F H HH NR⁸ Pyridinyl- — Methoxyphenyl- F H H H NR⁸ Methyl- — 5-Phenyl- Cl H HH [1,3,4]thiadiazol- 2-yl NR⁸ Butyryl- — 5-Phenyl- Cl H H H[1,3,4]thiadiazol- 2-yl NR⁸ Ethylcarbamoyl- — 5-Phenyl- Cl H H H[1,3,4]thiadiazol- 2-yl NR⁸ Ethoxycarbonyl- — 1H-imidazol-2yl Cl H H HNR⁸ Ethoxycarbonyl- — Isoxazol-3-yl Cl H H H NR⁸ Butyl- — 5-Phenyl- Cl HH H [1,3,4]thiadiazol- 2-yl NR⁸ Formyl- — 5-Phenyl- Cl H H H[1,3,4]thiadiazol- 2-yl NR⁸ Isobutyryl- — 5-Phenyl- Cl H H H[1,3,4]thiadiazol- 2-yl NR⁸ 2,3-Dihydroxypropionyl- — 5-Phenyl- Cl H H H[1,3,4]thiadiazol- 2-yl NR⁸ (1-hydroxypropan-2- — 5-Phenyl- Cl H H Hyloxy)carbonyl- [1,3,4]thiadiazol- 2-yl NR⁸ Ethoxycarbonyl- —Oxazol-2-yl Cl H H H NR⁸ Ethoxycarbonyl- — 2H-tetrazol-5-yl Cl H H H NR⁸t-Butoxycarbonyl- — 1H-imidazol-2-yl Cl H H H NR⁸ Acetyl- —1H-imidazol-2-yl Cl H H H NR⁸ Ethoxycarbonyl- — 4-Methyl-1H- Cl H H Himidazol-2-yl NR⁸ i-Propoxycarbonyl- — 1H-imidazol-2-yl Cl H H H NR⁸Methoxycarbonyl- — 1H-imidazol-2-yl Cl H H H NR⁸ Butyrl- —1H-imidazol-2-yl Cl H H H NR⁸ Dimethylaminocarbonyl- — 1H-imidazol-2-ylCl H H H NR⁸ Methoxyacetyl- — 1H-imidazol-2-yl Cl H H H NR⁸2-Methylpropan-1- — 1H-imidazol-2-yl Cl H H H oxycarbonyl NR⁸3-Methylbutyrl- — 1H-imidazol-2-yl Cl H H H NR⁸ 2,2-Dimethylpropan-1- —1H-imidazol-2-yl Cl H H H oxycarbonyl- NR⁸ Isobutyrl- — 1H-imidazol-2-ylCl H H H NR⁸ Ethylsulfonyl- — 1H-imidazol-2-yl Cl H H H NR⁸Butoxycarbonyl- — 1H-imidazol-2-yl Cl H H H NR⁸ Cyclohexyloxycarbonyl- —1H-imidazol-2-yl Cl H H H NR⁸ Cyclopentyloxycarbonyl- — 1H-imidazol-2-ylCl H H H NR⁸ Formyl- — 1H-imidazol-2-yl Cl H H H NR⁸ sec-butoxycarbonyl-— 1H-imidazol-2-yl Cl H H H (especially the S-isomer) NR⁸Piperidin-1-ylcarbonyl- — 1H-imidazol-2-yl Cl H H H NR⁸pyrrolidin-1-ylcarbonyl- — 1H-imidazol-2-yl Cl H H H NR⁸4-methylpiperazin-1- — 1H-imidazol-2-yl Cl H H H ylcarbonyl- NR⁸Diethylaminocarbonyl- — 1H-imidazol-2-yl Cl H H H NR⁸Ethylaminocarbonyl- — 1H-imidazol-2-yl Cl H H H NR⁸ Cyclohexylcarbonyl-— 1H-imidazol-2-yl Cl H H H NR⁸ 2-(tetrahydro-2H-pyran-4- —1H-imidazol-2-yl Cl H H H yl)acetyl- NR⁸ 2-(1-(tert- — 1H-imidazol-2-ylCl H H H butoxycarbonyl)piperidin-4- yl)acetyl- NR⁸2-(piperidin-4-yl)acetyl- — 1H-imidazol-2-yl Cl H H H NR⁸ Pentanoyl- —1H-imidazol-2-yl Cl H H H NR⁸ Cyclopropylacetyl- — 1H-imidazol-2-yl Cl HH H NR⁸ Propanoyl- — 1H-imidazol-2-yl Cl H H H NR⁸ 3,3-dimethylbutanoyl-— 1H-imidazol-2-yl Cl H H H NR⁸ Cyclopentylcarbonyl- — 1H-imidazol-2-ylCl H H H NR⁸ t-butoxycarbonyl- — 4,5-Dihydro-5- Cl H H H —oxo-1H-imidazol- 2-yl NR⁸ Cyclopropylcarbonyl- — 1H-imidazol-2-yl Cl H HH NR⁸ Ethoxyacetyl- — 1H-imidazol-2-yl Cl H H H NR⁸ Benzyloxyacetyl- —1H-imidazol-2-yl Cl H H H NR⁸ tetrahydrofuran-2-ylcarbonyl- —1H-imidazol-2-yl Cl H H H NR⁸ Dimethylaminosulfonyl- — 1H-imidazol-2-ylCl H H H NR⁸ N-(t-butoxycarbonyl) — 1H-imidazol-2-yl Cl H H Haminosulfonyl- NR⁸ Aminosulfonyl- — 1H-imidazol-2-yl Cl H H H NR⁸t-butoxycarbonyl- — Pyrazin-2-yl Cl H H H NR⁸ t-butoxycarbonyl- —Pyridinyl- Cl H H H NR⁸ t-butoxycarbonyl- — Methyl-pyridinyl- Cl H H HNR⁸ t-butoxycarbonyl- — Isoxazol-3-yl Cl H H H NR⁸ Methoxycarbonyl- —1H-imidazol-2-yl Cl H H H NR⁸ Acetyl- — Pyridinyl Cl H H H NR⁸Methoxycarbonyl- — Pyridinyl Cl H H H NR⁸ t-butoxycarbonyl- — 5-Phenyl-Cl H H H [1,3,4]thiadiazol- 2-yl NR⁸CH₂ 2-cyclohexylacetyl- —1H-imidazol-2yl Cl H H H NR⁸CH₂ sec-butoxycarbonyl- — 1H-imidazol-2yl ClH H H (especially the S-isomer) NR⁸CH₂ Ethoxycarbonyl- — 5-Phenyl- Cl HH H [1,3,4]thiadiazol- 2-yl NR⁸CH₂ Isobutyryl- — 5-Phenyl- Cl H H H[1,3,4]thiadiazol- 2-yl NR⁸CH₂ Methoxycarbonyl- — 5-Phenyl- Cl H H H[1,3,4]thiadiazol- 2-yl NR⁸CH₂ Isobutyryl- — 5-Phenyl- Cl H H H[1,3,4]thiadiazol- 2-yl NR⁸CH₂ Formyl- — 5-Phenyl- Cl H H H[1,3,4]thiadiazol- 2-yl O — — 5-Phenyl- Cl H H H [1,3,4]thiadiazol- 2-ylO — — 5-Phenyl- Cl H H H [1,3,4]thiadiazol- 2-yl O — — 1H-imidazol-2ylCl H H H O — — Thiazol-2-yl Cl H H H O — — Thiazol-2-yl Cl H H H O — —5-Phenyl- Cl H H H [1,3,4]thiadiazol- 2-yl O — — 5-(p-Chloro- Cl H H Hphenyl)- [1,3,4]thiadiazol- 2-yl O — — 5-Phenyl- F H H H[1,3,4]thiadiazol- 2-yl O — — 1H-imidazol-2yl Cl H H H O — —5-phenyl-1H- Cl H H H imidazol-2-yl O — — 5- Cl H H H phenylpyrimidin-2-yl S — — 1H-imidazol-2-yl Cl H H H

X R⁸ R³ R⁴ R⁵ R⁶ R⁷ CHR⁸ N-methyl-N-acetyl-amino- 1H-imidazol-2-yl Cl HH H CHR⁸ N-(t-butoxycarbonyl)-N- 1H-imidazol-2-yl Cl H H H methylamino-CHR⁸ Hydrogen 1H-imidazol-2-yl Cl H H H CHR⁸N-(Dimethylaminocarbonyl)-N- 5-Phenyl- Cl H H H methylamino-[1,3,4]thiadiazol- 2-yl CHR⁸ N-(3-methylbutyrl)-N- 5-Phenyl- Cl H H Hmethylamino- [1,3,4]thiadiazol- 2-yl CHR⁸ N-(Propoxycarbonyl)-N-5-Phenyl- Cl H H H methylamino- [1,3,4]thiadiazol- 2-yl CHR⁸N-(i-propoxycarbonyl)-N- 5-Phenyl- Cl H H H methylamino-[1,3,4]thiadiazol- 2-yl CHR⁸ N-butyryl-N-methylamino- 5-Phenyl- Cl H H H[1,3,4]thiadiazol- 2-yl CHR⁸ N-(ethoxycarbonyl)-N- 5-Phenyl- Cl H H Hmethylamino- [1,3,4]thiadiazol- 2-yl CHR⁸ N-(Ethoxycarbonyl)amino-5-Phenyl- Cl H H H [1,3,4]thiadiazol- 2-yl CHR⁸N-(isopropoxycarbonyl)amino- 5-Phenyl- Cl H H H [1,3,4]thiadiazol- 2-ylCHR⁸ N-(methoxycarbonyl)amino- 5-Phenyl- Cl H H H [1,3,4]thiadiazol-2-yl

More particularly, compounds of the invention include

X R⁸ R⁹ R³ R⁴ R⁵ R⁶ R⁷ CHR⁸ Methoxycarbonyl- H 5-Phenyl- Cl H H H[1,3,4]thiadiazol- 2-yl CR⁸R⁸ Methylenedioxy- 5-Phenyl- Cl H H H[1,3,4]thiadiazol- 2-yl CR⁹R⁸ Ethylenedioxy- 5-Phenyl- Cl H H H[1,3,4]thiadiazol- 2-yl NR⁸ Ethoxycarbonyl- — 5-Phenyl- Cl H H H[1,3,4]thiadiazol- 2-yl NR⁸ Acetyl- — 5-Phenyl- Cl H H H[1,3,4]thiadiazol- 2-yl NR⁸ Methoxycarbonyl- — 5-Phenyl- Cl H H H[1,3,4]thiadiazol- 2-yl NR⁸ Methoxyacetyl- — 5-Phenyl- Cl H H H[1,3,4]thiadiazol- 2-yl NR⁸ 3-Methylbutanoyl- — 5-Phenyl- Cl H H H[1,3,4]thiadiazol- 2-yl NR⁸ Propoxycarbonyl- — 5-Phenyl- Cl H H H[1,3,4]thiadiazol- 2-yl NR⁸ i-Propoxycarbonyl- — 5-Phenyl- Cl H H H[1,3,4]thiadiazol- 2-yl NR⁸ Dimethylaminocarbonyl- — 5-Phenyl- Cl H H H[1,3,4]thiadiazol- 2-yl NR⁸ Propoxycarbonyl- — 1H-imidazol-2yl Cl H H HNR⁸ sec-Butoxycarbonyl- — 1H-imadazol-2yl Cl H H H (especially the R-isomer) NR⁸ 2-Cyclopentylacetyl- — 1H-imidazol-2yl Cl H H H NR⁸2-Cyclohexylacetyl- — 1H-imidazol-2yl Cl H H H NR⁸ Butyrl- — 5-Phenyl-Cl H H H [1,3,4]thiadiazol- 2-yl NR⁸ Ethoxycarbonyl- —H 5-Phenyl- Cl H HH [1,3,4]thiadiazol- 2-yl NR⁸ Methoxyacetyl- — 5-Phenyl- Cl H H H[1,3,4]thiadiazol- 2-yl NR⁸ Acetyl- — 5-Phenyl- Cl H H H[1,3,4]thiadiazol- 2-yl NR⁸ Methyl- — 5-Phenyl- Cl H H H[1,3,4]thiadiazol- 2-yl NR⁸CH₂ Acetyl- — 5-Phenyl- Cl H H H[1,3,4]thiadiazol- 2-yl NR⁸CH₂ 2-Cyclohexylacetyl- — 1H-imidazol-2yl ClH H H NR⁸CH₂ sec-Butoxycarbonyl- — 1H-imidazol-2yl Cl H H H (especiallythe S- isomer) O — — 5-Phenyl- Cl H H H [1,3,4]thiadiazol- 2-yl O — —5-Phenyl- Cl H H H [1,3,4]thiadiazol- 2-yl O — — 1H-imidazol-2yl Cl H HH O — — Thiazol-2-yl Cl H H H

Utility, Testing and Administration

Utility

The compounds of the present invention are selective for and modulatethe cardiac sarcomere, and are useful to bind to and/or potentiate theactivity of cardiac myosin, increasing the rate at which myosinhydrolyzes ATP. As used in this context, “modulate” means eitherincreasing or decreasing myosin activity, whereas “potentiate” means toincrease activity. It has also been determined in testing representativecompounds of the invention, that their administration can also increasethe contractile force in cardiac muscle fiber.

The compounds, pharmaceutical formulations and methods of the inventionare used to treat heart disease, including but not limited to: acute (ordecompensated) congestive heart failure, and chronic congestive heartfailure; particularly diseases associated with systolic heartdysfunction. Additional therapeutic utilities include administration tostabilize heart function in patients awaiting a heart transplant, and toassist a stopped or slowed heart in resuming normal function followinguse of a bypass pump.

Testing

ATP hydrolysis is employed by myosin in the sarcomere to produce force.Therefore, an increase in ATP hydrolysis would correspond to an increasein the force or velocity of muscle contraction. In the presence ofactin, myosin ATPase activity is stimulated>100 fold. Thus, ATPhydrolysis not only measures myosin enzymatic activity but also itsinteraction with the actin filament. A compound that modulates thecardiac sarcomere can be identified by an increase or decrease in therate of ATP hydrolysis by myosin, preferably exhibiting a 1.4 foldincrease at concentrations less than 10 μM (more preferably, less than 1μM). Preferred assays for such activity will employ myosin from a humansource, although myosin from other organisms can also be used. Systemsthat model the regulatory role of calcium in myosin binding are alsopreferred.

Alternatively, a biochemically functional sarcomere preparation can beused to determine in vitro ATPase activity, for example, as described inU.S. Ser. No. 09/539,164, filed Mar. 29, 2000. The functionalbiochemical behavior of the sarcomere, including calcium sensitivity ofATPase hydrolysis, can be reconstituted by combining its purifiedindividual components (particularly including its regulatory componentsand myosin). Another functional preparation is the in vitro motilityassay. It can be performed by adding test compound to a myosin-boundslide and observing the velocity of actin filaments sliding over themyosin covered glass surface (Kron S J. (1991) Methods Enzymol.196:399-416).

The in vitro rate of ATP hydrolysis correlates to myosin potentiatingactivity, which can be determined by monitoring the production of eitherADP or phosphate, for example as described in Ser. No. 09/314,464, filedMay 18, 1999. ADP production can also be monitored by coupling the ADPproduction to NADH oxidation (using the enzymes pyruvate kinase andlactate dehydrogenase) and monitoring the NADH level either byabsorbance or fluorescence (Greengard, P., Nature 178 (Part 4534):632-634 (1956); Mol Pharmacol 1970 January; 6(1):31-40). Phosphateproduction can be monitored using purine nucleoside phosphorylase tocouple phosphate production to the cleavage of a purine analog, whichresults in either a change in absorbance (Proc Natl Acad Sci USA 1992Jun. 1; 89(11):4884-7) or fluorescence (Biochem J 1990 Mar. 1;266(2):611-4). While a single measurement can be employed, it ispreferred to take multiple measurements of the same sample at differenttimes in order to determine the absolute rate of the protein activity;such measurements have higher specificity particularly in the presenceof test compounds that have similar absorbance or fluorescenceproperties with those of the enzymatic readout.

Test compounds can be assayed in a highly parallel fashion usingmultiwell plates by placing the compounds either individually in wellsor testing them in mixtures. Assay components including the targetprotein complex, coupling enzymes and substrates, and ATP can then beadded to the wells and the absorbance or fluorescence of each well ofthe plate can be measured with a plate reader.

A preferred method uses a 384 well plate format and a 25 μL reactionvolume. A pyruvate kinase/lactate dehydrogenase coupled enzyme system(Huang T G and Hackney D D. (1994) J Biol Chem 269(23):16493-16501) isused to measure the rate of ATP hydrolysis in each well. As will beappreciated by those in the art, the assay components are added inbuffers and reagents. Since the methods outlined herein allow kineticmeasurements, incubation periods are optimized to give adequatedetection signals over the background. The assay is done in real timegiving the kinetics of ATP hydrolysis, which increases the signal tonoise ratio of the assay.

Modulation of cardiac muscle fiber contractile force can be measuredusing detergent permeabilized cardiac fibers (also referred to asskinned cardiac fibers), for example, as described by Haikala H, et al(1995) J Cardiovasc Pharmacol 25(5):794-801. Skinned cardiac fibersretain their intrinsic sarcomeric organization, but do not retain allaspects of cellular calcium cycling, this model offers two advantages:first, the cellular membrane is not a barrier to compound penetration,and second, calcium concentration is controlled. Therefore, any increasein contractile force is a direct measure of the test compound's effecton sarcomeric proteins. Tension measurements are made by mounting oneend of the muscle fiber to a stationary post and the other end to atransducer that can measure force. After stretching the fiber to removeslack, the force transducer records increased tension as the fiberbegins to contract. This measurement is called the isometric tension,since the fiber is not allowed to shorten. Activation of thepermeabilized muscle fiber is accomplished by placing it in a bufferedcalcium solution, followed by addition of test compound or control. Whentested in this manner, compounds of the invention caused an increase inforce at calcium concentrations associated with physiologic contractileactivity, but very little augmentation of force in relaxing buffer atlow calcium concentrations or in the absence of calcium (the EGTA datapoint).

Selectivity for the cardiac sarcomere and cardiac myosin can bedetermined by substituting non-cardiac sarcomere components and myosinin one or more of the above-described assays and comparing the resultsobtained against those obtained using the cardiac equivalents.

A compound's ability to increase observed ATPase rate in an in vitroreconstituted sarcomere assay could result from the increased turnoverrate of S1-myosin or, alternatively, increased sensitivity of adecorated actin filament to Ca⁺⁺-activation. To distinguish betweenthese two possible modes of action, the effect of the compound on ATPaseactivity of S1 with undecorated actin filaments is initially measured.If an increase of activity is observed, the compound's effect on theCa-responsive regulatory apparatus could be disproved. A second, moresensitive assay, can be employed to identify compounds whose activatingeffect on S1-myosin is enhanced in the presence of a decorated actin(compared to pure actin filaments). In this second assay activities ofcardiac-S1 and skeletal-S1 on cardiac and skeletal regulated actinfilaments (in all 4 permutations) are compared. A compound that displaysits effect on cardiac-S1/cardiac actin and cardiac-S1/skeletal actin,but not on skeletal-S1/skeletal actin and skeletal-S1/cardiac actinsystems, can be confidently classified as cardiac-S1 activator.

Initial evaluation of in vivo activity can be determined in cellularmodels of myocyte contractility, e.g., as described by Popping S, et al((1996) Am. J. Physiol. 271: H₃₅₇-H₃₆₄) and Wolska B M, et al ((1996)Am. J. Physiol. 39:H24-H32). One advantage of the myocyte model is thatthe component systems that result in changes in contractility can beisolated and the major site(s) of action determined. Compounds withcellular activity (for example, selecting compounds having the followingprofile: >120% increase in fractional shortening over basal at 2 μM,limited changes in diastolic length (<5% change), and no significantdecrease in contraction or relaxation velocities) can then be assessedin whole organ models, such as such as the Isolated Heart (Langendorff)model of cardiac function, in vivo using echocardiography or invasivehemodynamic measures, and in animal-based heart failure models, such asthe Rat Left Coronary Artery Occlusion model. Ultimately, activity fortreating heart disease is demonstrated in blinded, placebo-controlled,human clinical trials.

Administration

The compounds of Formula I are administered at a therapeuticallyeffective dosage, e.g., a dosage sufficient to provide treatment for thedisease states previously described. While human dosage levels have yetto be optimized for the compounds of the invention, generally, a dailydose is from about 0.05 to 100 mg/kg of body weight, preferably about0.10 to 10.0 mg/kg of body weight, and most preferably about 0.15 to 1.0mg/kg of body weight. Thus, for administration to a 70 kg person, thedosage range would be about 3.5 to 7000 mg per day, preferably about 7.0to 700.0 mg per day, and most preferably about 10.0 to 100.0 mg per day.The amount of active compound administered will, of course, be dependenton the subject and disease state being treated, the severity of theaffliction, the manner and schedule of administration and the judgmentof the prescribing physician; for example, a likely dose range for oraladministration would be about 70 to 700 mg per day, whereas forintravenous administration a likely dose range would be about 700 to7000 mg per day, the active agents being selected for longer or shorterplasma half-lives, respectively.

Administration of the compounds of the invention or the pharmaceuticallyacceptable salts thereof can be via any of the accepted modes ofadministration for agents that serve similar utilities including, butnot limited to, orally, subcutaneously, intravenously, intranasally,topically, transdermally, intraperitoneally, intramuscularly,intrapulmonarilly, vaginally, rectally, or intraocularly. Oral andparenteral administration are customary in treating the indications thatare the subject of the present invention.

Pharmaceutically acceptable compositions include solid, semi-solid,liquid and aerosol dosage forms, such as, e.g., tablets, capsules,powders, liquids, suspensions, suppositories, aerosols or the like. Thecompounds can also be administered in sustained or controlled releasedosage forms, including depot injections, osmotic pumps, pills,transdermal (including electrotransport) patches, and the like, forprolonged and/or timed, pulsed administration at a predetermined rate.Preferably, the compositions are provided in unit dosage forms suitablefor single administration of a precise dose.

The compounds can be administered either alone or more typically incombination with a conventional pharmaceutical carrier, excipient or thelike (e.g., mannitol, lactose, starch, magnesium stearate, sodiumsaccharine, talcum, cellulose, sodium crosscarmellose, glucose, gelatin,sucrose, magnesium carbonate, and the like). If desired, thepharmaceutical composition can also contain minor amounts of nontoxicauxiliary substances such as wetting agents, emulsifying agents,solubilizing agents, pH buffering agents and the like (e.g., sodiumacetate, sodium citrate, cyclodextrine derivatives, sorbitanmonolaurate, triethanolamine acetate, triethanolamine oleate, and thelike). Generally, depending on the intended mode of administration, thepharmaceutical formulation will contain about 0.005% to 95%, preferablyabout 0.5% to 50% by weight of a compound of the invention. Actualmethods of preparing such dosage forms are known, or will be apparent,to those skilled in this art; for example, see Remington'sPharmaceutical Sciences, Mack Publishing Company, Easton, Pa.

In addition, the compounds of the invention can be co-administered with,and the pharmaceutical compositions can include, other medicinal agents,pharmaceutical agents, adjuvants, and the like. Suitable additionalactive agents include, for example: therapies that retard theprogression of heart failure by down-regulating neurohormonalstimulation of the heart and attempt to prevent cardiac remodeling(e.g., ACE inhibitors or β-blockers); therapies that improve cardiacfunction by stimulating cardiac contractility (e.g., positive inotropicagents, such as the β-adrenergic agonist dobutamine or thephosphodiesterase inhibitor milrinone); and therapies that reducecardiac preload (e.g., diuretics, such as furosemide).

In one preferred embodiment, the compositions will take the form of apill or tablet and thus the composition will contain, along with theactive ingredient, a diluent such as lactose, sucrose, dicalciumphosphate, or the like; a lubricant such as magnesium stearate or thelike; and a binder such as starch, gum acacia, polyvinylpyrrolidine,gelatin, cellulose, cellulose derivatives or the like. In another soliddosage form, a powder, marume, solution or suspension (e.g., inpropylene carbonate, vegetable oils or triglycerides) is encapsulated ina gelatin capsule.

Liquid pharmaceutically administrable compositions can, for example, beprepared by dissolving, dispersing, etc. an active compound as definedabove and optional pharmaceutical adjuvants in a carrier (e.g., water,saline, aqueous dextrose, glycerol, glycols, ethanol or the like) toform a solution or suspension. Injectables can be prepared inconventional forms, either as liquid solutions or suspensions, asemulsions, or in solid forms suitable for dissolution or suspension inliquid prior to injection. The percentage of active compound containedin such parenteral compositions is highly dependent on the specificnature thereof, as well as the activity of the compound and the needs ofthe subject. However, percentages of active ingredient of 0.01% to 10%in solution are employable, and will be higher if the composition is asolid which will be subsequently diluted to the above percentages.Preferably the composition will comprise 0.2-2% of the active agent insolution.

Formulations of the active compound or a salt may also be administeredto the respiratory tract as an aerosol or solution form nebulizer, or asa microfine powder for insufflation, alone or in combination with aninert carrier such as lactose. In such a case, the particles of theformulation have diameters of less than 50 microns, preferably less than10 microns.

Use in Screening

Generally, to employ the compounds of the invention in a method ofscreening for myosin binding, myosin is bound to a support and acompound of the invention is added to the assay. Alternatively, thecompound of the invention can be bound to the support and the myosinadded. Classes of compounds among which novel binding agents may besought include specific antibodies, non-natural binding agentsidentified in screens of chemical libraries, peptide analogs, etc. Ofparticular interest are screening assays for candidate agents that havea low toxicity for human cells. A wide variety of assays may be used forthis purpose, including labeled in vitro protein-protein binding assays,electrophoretic mobility shift assays, immunoassays for protein binding,functional assays (phosphorylation assays, etc.) and the like. See,e.g., U.S. Pat. No. 6,495,337, incorporated herein by reference.

EXAMPLES

The following examples serve to more fully describe the manner of usingthe above-described invention, as well as to set forth the best modescontemplated for carrying out various aspects of the invention. It isunderstood that these examples in no way serve to limit the true scopeof this invention, but rather are presented for illustrative purposes.All references cited herein are incorporated by reference in theirentirety.

Example 1

Preparation of4-(3-Carboxy-4-chloro-benzenesulfonyl)-piperazine-1-carboxylic acidethyl ester

2-Chloro-5-fluorosulfonylbenzoic acid (Acros Organics, 105 mg, 440 μmol,1.0 eq) was placed in a vial and the vial was flushed with nitrogen anda positive pressure was maintained. Anhydrous dichloromethane (1.4 mL)was added, followed by ethyl 1-piperazine carboxylate (80 μL, 528 μmol,1.2 eq) and ethyldiisopropylamine (redistilled, 80 μL, 440 μmol, 1.0eq). The mixture was stirred for 1.5 h after which time additionalpiperazine was added (60 μL, 352 μmol, 0.8 eq). After 14 h the mixturewas analyzed by reverse-phase HPLC-MS in negative ionization mode. Thesulfonyl fluoride starting material was present, as well as a less polarcompound showing (M−1) ion in about 35:65 ratio. An additional 0.35 eqof piperazine and ethyldiisopropylamine each (154 μmol, 30 μl) wereadded and the mixture was stirred for 4 h. HPLC-MS at this timeindicated little progress and an additional 0.25 eq of piperazine (100μmol, 20 μL) was added. After stirring for 14 h HPLC-MS indicated thereaction was complete. The reaction mixture was diluted with 2 mL ofethyl acetate, washed with 1M HCl solution (2×1 mL) and dried overanhydrous sodium sulfate. The solution was filtered, the solventsremoved on a rotary evaporator and vacuum pump to afford 134 mg of awhite solid, 81%. NOTE: Addition of excess nucleophilic amine at thebeginning of the reaction results in significant bis addition, givingsulfonamide and carboxamide product. Stepwise addition of nucleophilicamine as needed suppresses formation of this side product.

Preparation of4-[4-Chloro-3-(5-phenyl-[1,3,4]thiadiazol-2-ylcarbamoyl)-benzenesulfonyl]-piperazine-1-carboxylicacid ethyl ester

The benzoic acid piperazine ethyl ester from above (131 mg, 348 μmol,1.0 eq) was placed in a vial along with2-amino-5-phenyl-1,3,4-thiadiazole sulfate (115 mg, 417 μmol, 1.2 eq),HBTU (Advanced Chem Tech, 198 mg, 521 μmol, 1.5 eq) and HOBt hydrate (80mg, 521 μmol, 1.5 eq). The vial was flushed with nitrogen and a positivepressure was maintained. Anhydrous dimethylformamide (1.7 mL) was added,followed by ethyldiisopropylamine (redistilled, 120 μL, 695 μmol, 2.0eq) and the mixture was stirred for 14 h. Analysis by reverse-phaseHPLC-MS in positive mode indicated that the acid starting material hadbeen consumed and replaced with a much less polar compound showing thedesired (M+1). The reaction mixture was diluted with 4 mL of water, theresulting precipitate was collected by filtration and washed with thefollowing: water×2, 1M HCl solution×2, saturated sodium bicarbonatesolution×2, water×2 and hexanes×1 and dried under suction. 119 mg of anoff-white solid was obtained, 64% yield.

Example 2 Preparation of4-(3-Carboxy-4-chloro-benzenesulfonyl)-piperazine-1-carboxylic acidtert-butyl ester

Prepared as for the compound above with these modifications: after theaddition of 2.0 eq total of tert-butyl 1-piperazine carboxylate on day1, HPLC-MS analysis showed the reaction to be only ⅓ complete. Anadditional 1.4 eq of piperazine was added and the reaction was stirredfor 24 h. After this time the reaction was found to be complete and theworkup was performed as for the ethyl case. Instead of washing with 1MHCl solution, 0.3M potassium hydrogen sulfate solution was used and awash with saturated sodium chloride solution was also added prior todrying and concentrating. A quantitative yield of desired product wasobtained.

Preparation of4-[4-Chloro-3-(1H-imidazol-2-ylcarbamoyl)-benzenesulfonyl]-piperazine-1-carboxylicacid tert-butyl ester

The benzoic acid piperazine tert-butyl ester from above (1.57 g, 3.88mmol, 1.0 eq) was placed in a 100 mL round-bottom flask along with2-aminoimidazole sulfate (Aldrich, 615 mg, 4.65 mmol, 1.2 eq), HATU (PEBiosystems, 2.21 g, 5.82 mmol, 1.5 eq) and HOAt (Avocado, 792 mg, 5.82mmol, 1.5 eq). The flask was capped with a septum, flushed with nitrogenand a positive pressure was maintained. Anhydrous dimethylformamide (17mL) was added, followed by ethyldiisopropylamine (redistilled, 1.4 mL,7.76 mmol, 2.0 eq) and the mixture was stirred for 14 h. Analysis byreverse-phase HPLC-MS in positive mode indicated that the acid startingmaterial had been consumed and replaced with a much less polar compoundshowing the desired (M+1). The reaction mixture was diluted with 80 mLof water, the resulting precipitate was collected by filtration andwashed with the following: water×3, hexanes×2 and dried under suction toafford 1.46 g of an off-white solid. The crude material was purified(silica gel flash column, 5.1 cm×15 cm) eluting withEtOAc-hexanes-triethylamine (89:10:1 v/v) then EtOAc to provide 896 mgoff-white solid, 49% yield. TLC EtOAc-triethylamine (99:1 v/v)R_(f)=0.27.

Preparation of2-Chloro-5-[4-(2-cyclohexyl-acetyl)-piperazine-1-sulfonyl]-N-(1H-imidazol-2-yl)-benzamide

The Boc-piperazine imidazole compound from above (109 mg, 232 μmol, 1.0eq) was placed in a vial and treated with 2.6 mL of 50:49:1trifluoroacetic acid/dichloromethane/triethylsilane. After 15 min Bocremoval was complete as evidenced by reverse-phase HPLC-MS, and thesolvents were removed in vacuo. The residue was azeotroped 3×chloroformand placed under vacuum for 1 h.1-ethyl-(3-dimethylaminopropyl)carbodiimide HCl (EDC) (Advanced ChemTech, 67 mg, 348 μmol, 1.5 eq), HOBt hydrate (57 mg, 371 μmol, 1.6 eq)were added to the vial, the vial was capped, flushed with nitrogen and apositive pressure was maintained. Anhydrous dichloromethane (1.7 mL) wasadded, followed by ethyldiisopropylamine (redistilled, 250 μL, 1.39mmol, 6.0 eq) and cyclohexylacetic acid (TCI, 40 μL, 278 μmol, 1.2 eq)and the mixture was stirred for 14 h. Analysis by reverse-phase HPLC-MSin positive mode indicated that the piperazine starting material hadbeen consumed and replaced with a much less polar compound showing thedesired (M+1). The reaction mixture was diluted with 4 mL of EtOAc, andthe organic layer was washed with 2 mL each of the following: water×3,saturated sodium bicarbonate solution×1, saturated sodium chloride×1.The organic extracts were dried over anhydrous sodium sulfate, filteredand concentrated to afford 61 mg of an off-white solid, 78% yield.

Preparation of4-[4-Chloro-3-(1H-imidazol-2-ylcarbamoyl)-benzenesulfonyl]-piperazine-1-carboxylicacid (R)-sec-butyl ester

The Boc-piperazine imidazole compound from above (148 mg, 315 μmol, 1.0eq) was placed in a vial and treated with 3.6 mL of 50:49:1trifluoroacetic acid/dichloromethane/triethylsilane as described abovefor the cyclohexyl acetamide compound. (R)-Carbonic acid sec-butyl ester4-nitro-phenyl ester, 93% wt. (89 mg, 346 μmol, 1.1 eq) and 3 mg DMAPwere added to the vial, the vial was capped, flushed with nitrogen and apositive pressure was maintained. Anhydrous DMF (2.1 mL) was added,followed by ethyldiisopropylamine (redistilled, 170 μL, 945 μmol, 3.0eq) and the mixture was stirred for 14 h at 40° C. Analysis byreverse-phase HPLC-MS in positive mode indicated that the very polarunprotected piperazine starting material had been consumed and replacedwith a much less polar compound showing the desired (M+1), as well as asmall amount of even less polar bis-acylated material. The heat wasremoved and the reaction mixture was diluted with 4 mL of 2N NaOHsolution and stirred for 10 min. The mixture was extracted with EtOAc(3×2 mL), and the organic layer was washed with 2 mL each of thefollowing: 2N NaOH×5 (until yellow color disappeared), water×1, 10% HOAcsolution×1, water×1, saturated sodium bicarbonate solution×1, saturatedsodium chloride×1. The organic extracts were dried over anhydrous sodiumsulfate, filtered and concentrated to afford 121 mg of an off-whitesolid, 82% yield.

Preparation of4-[4-Chloro-3-(1H-imidazol-2-ylcarbamoyl)-benzenesulfonyl]-piperazine-1-carboxylicacid iso-propyl ester

The Boc-piperazine imidazole compound from above (86 mg, 183 μmol, 1.0eq) was placed in a vial and treated with 1.8 mL of 50:49:1trifluoroacetic acid/dichloromethane/triethylsilane as described abovefor the cyclohexyl acetamide compound. DMAP (2 mg) were added to thevial, the vial was capped, flushed with nitrogen and a positive pressurewas maintained. Anhydrous DCM (2.1 mL) was added, followed byethyldiisopropylamine (redistilled, 120 μL, 640 μmol, 3.5 eq) and a 1.0M solution of isopropyl chloroformate in toluene (220 μL, 220 μmol, 1.2eq) and the mixture was stirred for 14 h. Analysis by reverse-phaseHPLC-MS in positive mode indicated that the very polar unprotectedpiperazine starting material had been consumed and replaced with a muchless polar compound showing the desired (M+1). A solid had precipitated.The solvent was removed in vacuo and the solid was taken up in 1 mL ofDMF and re-precipitated with 4 mL of water, isolated by filtration andwashed with: water×3, hexanes×2 and dried under suction. A white solidwas obtained, 50 mg (60% yield).

Example 3

Preparation of4-[4-Chloro-3-(1H-imidazol-2-ylcarbamoyl)-benzenesulfonyl]-N-cyano-piperazine-1-carboximidicacid phenyl ester

The Boc-piperazine imidazole compound from above (2.04 g, 4.33 mmol, 1.0eq) was treated with 50:49:1 trifluoroaceticacid/dichloromethane/triethylsilane as described above for thecyclohexyl acetamide compound. The residue was treated with saturatedsodium bicarbonate solution until pH>8, then the solution was extractedwith EtOAc and concentrated in vacuo to afford 1.6 g free amine.Diphenylcyanocarbonimidate (1.08 g, 4.54 mmol, 10.5 eq) was added to theflask and it was capped, flushed with nitrogen and a positive pressurewas maintained. Anhydrous THF (30 mL) was added, followed byethyldiisopropylamine (redistilled, 830 μL, 4.76 mmol, 1.1 eq) and themixture was stirred for 1 h. Analysis by reverse-phase HPLC-MS inpositive mode indicated that the very polar unprotected piperazinestarting material had been consumed and replaced with a compound showingthe desired (M+1). The solvents were removed in vacuo.

Preparation of2-Chloro-5-[4-(N-cyclopentyl-N′-cyano-carbamimidoyl)-piperazine-1-sulfonyl]-N-(1H-imidazol-2-yl)-benzamide

The phenyl imidate from above (100 mg, 195 μmol, 1.0 eq) was placed in avial which was capped, flushed with nitrogen and a positive pressure wasmaintained. Anhydrous THF (5 mL) was added, followed by cyclopentylamine(100 μL, 973 μmol, 5.0 eq) and the mixture was stirred for 48 h afterwhich the solvent was removed in vacuo. The residue was purified byreverse-phase HPLC to afford 27 mg product, 27% yield.

Example 4 Preparation of2-Chloro-5-(4-methyl-piperazine-1-sulfonyl)-benzoic acid

2-Chloro-5-fluorosulfonylbenzoic acid (Acros Organics, 195 mg, 813 1.0eq) was placed in a vial and the vial was flushed with nitrogen and apositive pressure was maintained. Anhydrous dichloromethane (2.7 mL) wasadded, followed by 1-methylpiperazine (110 μL, 976 μmol, 1.2 eq). Themixture was stirred for 2 h after which time an additional 1.2 eq wasadded. After 14 h the mixture was analyzed by reverse-phase HPLC-MS inpositive ionization mode. The less polar sulfonyl fluoride startingmaterial was gone and replaced with a very polar compound with thedesired (M+1). The solvents were removed in vacuo to afford 407 mg of apale yellow solid. By ¹H-NMR the desired compound was present,contaminated with methylpiperazine.

Preparation of 2-Chloro-5-(4-methyl-piperazine-1-sulfonyl)-benzoic acidmethyl ester

The crude methylpiperazine benzoic acid from above (259 mg, 813 μmol,1.0 eq) was dissolved in 4 mL of 30% MeOH in benzene. 2.0 MTrimethylsilyl diazomethane in hexanes solution (410 μL, 813 μmol, 1.0eq) was added dropwise. After 15 min the mixture was analyzed byreverse-phase HPLC-MS in positive mode. Starting material was present,as well as less polar product. Another 1.0 eq of diazomethane compoundwas added and the reaction was monitored again after 15 min. Anadditional 0.2 eq of diazomethane was added (90 μL, 178 μmol) and after15 min the reaction was judged to be complete by HPLC-MS. A few dropsglacial HOAc were added until the yellow color disappeared and thesolvents were removed in vacuo to afford 491 mg of a yellow glass. Thiswas taken up in 2 mL 50%-saturated sodium bicarbonate solution (pH>8)and extracted with dichloromethane (3×1 mL). The extracts were driedover anhydrous sodium sulfate, filtered and concentrated to afford 224mg of yellow oil, 83%.

Preparation of Lithium 2-chloro-5-(4-methyl-piperazine-1-sulfonyl)-benzoate

The methylpiperazine methyl ester from above (197 mg, 592 μmol, 1.0 eq)was placed in a vial, dissolved in 1.5 mL of MeOH and 30 μL of water.LiOH hydrate (26 mg, 622 μmol, 1.05 eq) was added and the vial wascapped, flushed with nitrogen and a positive pressure was maintained.The mixture was heated at 60° C. for 4 h at which time it was judged tobe complete by reverse-phase HPLC-MS. The solvents were removed in vacuoto afford 197 mg of a white solid.

Preparation of2-Chloro-5-(4-methyl-piperazine-1-sulfonyl)-N-(5-phenyl-[1,3,4]thiadiazol-2-yl)-benzamide

The methylpiperazine acid salt from above (93 mg, 286 μmol, 1.0 eq) wascoupled with 2-amino-5-phenyl-1,3,4-thiadiazole sulfate using the HBTUprotocol as described above, except that the ethyldiisopropylamine wasadded 1 h after the reaction was started in order to neutralize the acidsalt and effect dissolution. After 3 h total the reaction was completeby reverse-phase HPLC-MS analysis in positive mode. The reaction mixturewas diluted with 3 mL of water and taken to pH=8 by addition ofsaturated bicarbonate solution. The resulting precipitate was collectedby filtration and washed with the following: water×2 and hexanes×1. Theproduct was isolated as 109 mg of an off-white solid, 80% yield.

Example 5

Synthesis of Starting Materials

2-Amino-4-phenylimidazole, a reagent in the synthesis of compounds ofFormula I, was synthesized from the procedure of: Little, T. L.; Webber,S. E. “A Simple and Practical Synthesis of 2-Aminoimidazoles” J. Org.Chem. 1994, 59, 7299-7305, which is incorporated herein by reference.

2-Amino-5-phenylpyrimidine was synthesized in an analogous fashion tothe procedure described in: Gong, Y.; Pauls, H. W. “A ConvenientSynthesis of Heteroarylbenzoic Acids via Suzuki Reaction” Synlett, 2000,6, 829-831, which is incorporated herein by reference.

2-Aminooxazole was prepared from the procedure of: Cockerill, A. F.;Deacon, A.; Harrison, R. G.; Osborne, D. J.; Prime, D. M.; Ross, W. J.;Todd, A.; Verge, J. P. “An Improved Synthesis of 2-Amino-1,3-OxazolesUnder Basic Conditions” Synthesis, 1976, 591-593, which is incorporatedherein by reference.

2-Amino-4-methylimidazole was prepared according to the proceduredescribed in: Little, T. L.; Webber, S. E. “A Simple and PracticalSynthesis of 2-Aminoimidazoles” J. Org. Chem. 1994, 59, 7299-7305, whichis incorporated herein by reference.

Example 6 Preparation of (R)-Carbonic acid sec-butyl ester4-nitro-phenyl ester

4-Nitrophenyl chloroformate (1.91 g, 9.50 mmol, 1.1 eq) was placed in avial and the vial was flushed with nitrogen and a positive pressure wasmaintained. Anhydrous dichloromethane (10 mL) was added, followed by(R)-sec-butyl alcohol (Acros Organics, 800 μL, 8.63 mmol, 1.0 eq) andanhydrous pyridine (770 μL, 9.50 mmol, 1.1 eq). After 15 h one nonpolarcompound was observed by HPLC-MS but which did not ionize in eitherpositive or negative mode. The reaction mixture was diluted with 10 mLof ethyl acetate, washed with 7 mL each of the following: 0.5 M NaOHsolution×2, water×1, 1M HCl solution×1, water×1, saturated sodiumchloride×1 and dried over anhydrous sodium sulfate. The solution wasfiltered, the solvents removed on a rotary evaporator and vacuum pump toafford 2.11 g of a pale yellow solid. This was shown to contain 11 mol %4-nitrophenol by ¹H-NMR and was judged to be 93% pure by weight. Totalyield was therefore 95%.

Example 7

Target Identification Assays

Specificity assays: Compound specificity towards cardiac myosin isevaluated by comparing the effect of the compound on actin-stimulatedATPase of a panel of myosin isoforms: cardiac, skeletal and smoothmuscle, at a single 50 μM compound concentration.

Myofibril assays: To evaluate the effect of compounds on the ATPaseactivity of full-length cardiac myosin in the context of nativesarcomere, skinned myofibril assays are performed. Rat cardiacmyofibrils are obtained by homogenizing rat cardiac tissue in thepresence of detergent. Such treatment removes membranes and majority ofsoluble cytoplasmic proteins but leaves intact cardiac sarcomericacto-myosin apparatus. Myofibril preparations retain the ability tohydrolyze ATP in an Ca⁺⁺ controlled manner. ATPase activities of suchmyofibril preparations in the presence and absence of compounds areassayed at Ca⁺⁺ concentrations giving 50% and 100% of a maximal rate.

Example 8

In vitro Model of Dose Dependent Cardiac Myosin ATPase Modulation

Dose responses are measured using a calcium-buffered, pyruvate kinaseand lactate dehydrogenase-coupled ATPase assay containing the followingreagents (concentrations expressed are final assay concentrations):Potassium PIPES (12 mM), MgCl₂ (2 mM), ATP (1 mM), DTT (1 mM), BSA (0.1mg/ml), NADH (0.5 mM), PEP (1.5 mM), pyruvate kinase (4 U/ml), lactatedehydrogenase (8 U/ml), and antifoam (90 ppm). The pH is adjusted to6.80 at 22° C. by addition of potassium hydroxide. Calcium levels arecontrolled by a buffering system containing 0.6 mM EGTA and varyingconcentrations of calcium, to achieve a free calcium concentration of1×10⁻⁴ M to 1×10⁻⁸ M.

The protein components specific to this assay are bovine cardiac myosinsubfragment-1 (typically 0.5 μM), bovine cardiac actin (14 μM), bovinecardiac tropomyosin (typically 3 μM), and bovine cardiac troponin(typically 3-8 μM). The exact concentrations of tropomyosin and troponinare determined empirically, by titration to achieve maximal differencein ATPase activity when measured in the presence of 1 mM EGTA versusthat measured in the presence of 0.2 mM CaCl₂. The exact concentrationof myosin in the assay is also determined empirically, by titration toachieve a desired rate of ATP hydrolysis. This varies between proteinpreparations, due to variations in the fraction of active molecules ineach preparation.

Compound dose responses are typically measured at the calciumconcentration corresponding to 50% of maximal ATPase activity (pCa₅₀),so a preliminary experiment is performed to test the response of theATPase activity to free calcium concentrations in the range of 1×10⁻⁴ Mto 1×10⁻⁸ M. Subsequently, the assay mixture is adjusted to the pCa₅₀(typically 3×10⁻⁷ M). Assays are performed by first preparing a dilutionseries of test compound, each with an assay mixture containing potassiumPipes, MgCl₂, BSA, DTT, pyruvate kinase, lactate dehydrogenase, myosinsubfragment-1, antifoam, EGTA, CaCl₂, and water. The assay is started byadding an equal volume of solution containing potassium Pipes, MgCl₂,BSA, DTT, ATP, NADH, PEP, actin, tropomyosin, troponin, antifoam, andwater. ATP hydrolysis is monitored by absorbance at 340 nm. Theresulting dose response curve is fit by the 4 parameter equationy=Bottom+((Top−Bottom)/(1+((EC50/X)^Hill))). The AC1.4 is defined as theconcentration at which ATPase activity is 1.4-fold higher than thebottom of the dose curve.

Example 9

Myocyte Assays

Preparation of adult cardiac ventricular rat myocytes. Adult maleSprague-Dawley rats are anesthetized with a mixture of isoflurane gasand oxygen. Hearts are quickly excised, rinsed and the ascending aortacannulated. Continuous retrograde perfusion is initiated on the heartsat a perfusion pressure of 60 cm H₂0. Hearts are first perfused with anominally Ca²⁺ free modified Krebs solution of the followingcomposition: 110 mM NaCl, 2.6 mM KCL, 1.2 mM KH₂PO₄ 7 H₂0, 1.2 mM MgSO₄,2.1 mM NaHCO₃, 11 mM glucose and 4 mM Hepes (all Sigma). This medium isnot recirculated and is continually gassed with O₂. After approximately3 minutes the heart is perfused with modified Krebs buffer supplementedwith 3.3% collagenase (169 μ/mg activity, Class II, WorthingtonBiochemical Corp., Freehold, N.J.) and 25 μM final calcium concentrationuntil the heart becomes sufficiently blanched and soft. The heart isremoved from the cannulae, the atria and vessels discarded and theventricles are cut into small pieces. The myocytes are dispersed bygentle agitation of the ventricular tissue in fresh collagenasecontaining Krebs prior to being gently forced through a 200 μm nylonmesh in a 50 cc tube. The resulting myocytes are resuspended in modifiedKrebs solution containing 25 μm calcium. Myocytes are made calciumtolerant by addition of a calcium solution (100 mM stock) at 10 minuteintervals until 100 μM calcium is achieved. After 30 minutes thesupernatant is discarded and 30-50 ml of Tyrode buffer (137 mM NaCL, 3.7mM KCL, 0.5 mM MgCL, 11 mM glucose, 4 mM Hepes, and 1.2 mM CaCl₂, pH7.4) is added to cells. Cells are kept for 60 min at 37° C. prior toinitiating experiments and used within 5 hrs of isolation. Preparationsof cells are used only if cells first passed QC criteria by respondingto a standard (>150% of basal) and isoproterenol (ISO; >250% of basal).Additionally, only cells whose basal contractility is between 3 and 8%are used in the following experiments.

Adult ventricular myocyte contractility experiments. Aliquots of Tyrodebuffer containing myocytes are placed in perfusion chambers (series 20RC-27NE; Warner Instruments) complete with heating platforms. Myocytesare allowed to attach, the chambers heated to 37° C., and the cells thenperfused with 37° C. Tyrode buffer. Myocytes are field stimulated at 1Hz in with platinum electrodes (20% above threshold). Only cells thathave clear striations, and are quiescent prior to pacing are used forcontractility experiments. To determine basal contractility, myocytesare imaged through a 40× objective and using a variable frame rate(60-240 Hz) charge-coupled device camera, the images are digitized anddisplayed on a computer screen at a sampling speed of 240 Hz. [Framegrabber, myopacer, acquisition, and analysis software for cellcontractility are available from IonOptix (Milton, Mass.).] After aminimum 5 minute basal contractility period, test compounds (0.01-15 μM)are perfused on the myocytes for 5 minutes. After this time, freshTyrode buffer is perfused to determine compound washout characteristics.Using edge detection strategy, contractility of the myocytes andcontraction and relaxation velocities are continuously recorded.

Contractility analysis: Three or more individual myocytes are tested percompound, using two or more different myocyte preparations. For eachcell, twenty or more contractility transients at basal (defined as 1 minprior to compound infusion) and after compound addition, are averagedand compared. These average transients are analyzed to determine changesin diastolic length, and using the Ionwizard analysis program(IonOptix), fractional shortening (% decrease in the diastolic length),and maximum contraction and relaxation velocities (um/sec) aredetermined. Analysis of individual cells are combined. Increase infractional shortening over basal indicates potentiation of myocytecontractility.

Calcium transient analysis: Fura loading: Cell permeable Fura-2(Molecular Probes) is dissolved in equal amounts of pluronic (MolProbes) and FBS for 10 min at RT. A 1 μM Fura stock solution is made inTyrode buffer containing 500 mM probenecid (Sigma). To load cells, thissolution is added to myocytes at RT. After 10 min. the buffer isremoved, the cells washed with Tyrode containing probenecid andincubated at RT for 10 min. This wash and incubation is repeated.Simultaneous contractility and calcium measurements are determinedwithin 40 min. of loading.

Imaging: A test compound is perfused on cells. Simultaneouscontractility and calcium transient ratios are determined at baselineand after compound addition. Cells are digitally imaged andcontractility determined as described above, using that a red filter inthe light path to avoid interference with fluorescent calciummeasurements. Acquisition, analysis software and hardware for calciumtransient analysis are obtained from IonOptix. The instrumentation forfluorescence measurement includes a xenon arc lamp and a Hyperswitchdual excitation light source that alternates between 340 and 380wavelengths at 100 Hz by a galvo-driven mirror. A liquid filled lightguide delivers the dual excitation light to the microscope and theemission fluorescence is determined using a photomultiplier tube (PMT).The fluorescence system interface routes the PMT signal and the ratiosare recorded using the IonWizard acquisition program.

Analysis: For each cell, ten or more contractility and calcium ratiotransients at basal and after compound addition, where averaged andcompared. Contractility average transients are analyzed using theIonwizard analysis program to determine changes in diastolic length, andfractional shortening (% decrease in the diastolic length). The averagedcalcium ratio transients are analyzed using the Ionwizard analysisprogram to determine changes in diastolic and systolic ratios and the75% time to baseline (T₇₅).

Durability: To determine the durability of response, myocytes arechallenged with a test compound for 25 minutes followed by a 2 min.washout period. Contractility response is compared at 5 and 25 min.following compound infusion.

Threshold potential: Myocytes are field stimulated at a voltageapproximately 20% above threshold. In these experiments the thresholdvoltage (minimum voltage to pace cell) is empirically determined, thecell paced at that threshold and then the test compound is infused.After the compound activity is at steady state, the voltage is decreasedfor 20 seconds and then restarted. Alteration of ion channelscorresponds to increasing or lowering the threshold action potential.

Hz frequency: Contractility of myocytes is determined at 3 Hz asfollows: a 1 min. basal time point followed by perfusion of the testcompound for 5 min. followed by a 2 min. washout. After the cellcontractility has returned completely to baseline the Hz frequency isdecreased to 1. After an initial acclimation period the cell ischallenged by the same compound. As this species, rat, exhibits anegative force frequency at 1Hz, at 3 Hz the FS of the cell should belower, but the cell should still respond by increasing its fractionalshortening in the presence of the compound.

Additive WITH Isoproterenol: To demonstrate that a compound act via adifferent mechanism than the adrenergic stimulant isoproterenol, cellsare loaded with fura-2 and simultaneous measurement of contractility andcalcium ratios are determined. The myocytes are sequentially challengedwith 5 μm a test compound, buffer, 2 nM isoproterenol, buffer, and acombination of a test compound and isoproterenol.

Example 10

In vitro Model of Dose Dependent Cardiac Myosin ATPase Modulation

Bovine and rat cardiac myosins are purified from the respective cardiactissues. Skeletal and smooth muscle myosins used in the specificitystudies are purified from rabbit skeletal muscle and chicken gizzards,respectively. All myosins used in the assays are converted to asingle-headed soluble form (S1) by a limited proteolysis withchymotrypsin. Other sarcomeric components: troponin complex, tropomyosinand actin are purified from bovine hearts (cardiac sarcomere) or chickenpectoral muscle (skeletal sarcomere).

Activity of myosins is monitored by measuring the rates of hydrolysis ofATP. Myosin ATPase is very significantly activated by actin filaments.ATP turnover is detected in a coupled enzymatic assay using pyruvatekinase (PK) and lactate dehydrogenase (LDH). In this assay each ADPproduced as a result of ATP hydrolysis is recycled to ATP by PK with asimultaneous oxidation of NADH molecule by LDH. NADH oxidation can beconveniently monitored by decrease in absorbance at 340 nm wavelength.

Dose responses are measured using a calcium-buffered, pyruvate kinaseand lactate dehydrogenase-coupled ATPase assay containing the followingreagents (concentrations expressed are final assay concentrations):Potassium PIPES (12 mM), MgCl₂ (2 mM), ATP (1 mM), DTT (1 mM), BSA (0.1mg/ml), NADH (0.5 mM), PEP (1.5 mM), pyruvate kinase (4 U/ml), lactatedehydrogenase (8 U/ml), and antifoam (90 ppm). The pH is adjusted to6.80 at 22° C. by addition of potassium hydroxide. Calcium levels arecontrolled by a buffering system containing 0.6 mM EGTA and varyingconcentrations of calcium, to achieve a free calcium concentration of1×10⁻⁴ M to 1×10⁻⁴ M.

The protein components specific to this assay are bovine cardiac myosinsubfragment-1 (typically 0.5 μM), bovine cardiac actin (14 μM), bovinecardiac tropomyosin (typically 3 μM), and bovine cardiac troponin(typically 3-8 μM). The exact concentrations of tropomyosin and troponinare determined empirically, by titration to achieve maximal differencein ATPase activity when measured in the presence of 1 mM EGTA versusthat measured in the presence of 0.2 mM CaCl₂. The exact concentrationof myosin in the assay is also determined empirically, by titration toachieve a desired rate of ATP hydrolysis. This varies between proteinpreparations, due to variations in the fraction of active molecules ineach preparation.

Compound dose responses are typically measured at the calciumconcentration corresponding to 50% of maximal ATPase activity (pCa₅₀),so a preliminary experiment is performed to test the response of theATPase activity to free calcium concentrations in the range of 1×10⁻⁴ Mto 1×10⁻⁸ M. Subsequently, the assay mixture is adjusted to the pCa₅₀(typically 3×10⁻⁷ M). Assays are performed by first preparing a dilutionseries of test compound, each with an assay mixture containing potassiumPipes, MgCl₂, BSA, DTT, pyruvate kinase, lactate dehydrogenase, myosinsubfragment-1, antifoam, EGTA, CaCl₂, and water. The assay is started byadding an equal volume of solution containing potassium Pipes, MgCl₂,BSA, DTT, ATP, NADH, PEP, actin, tropomyosin, troponin, antifoam, andwater. ATP hydrolysis is monitored by absorbance at 340 nm. Theresulting dose response curve is fit by the 4 parameter equationy=Bottom+((Top−Bottom)/(1+((EC50/X)^Hill))). The AC1.4 is defined as theconcentration at which ATPase activity is 1.4-fold higher than thebottom of the dose curve.

Ability of a compound to activate cardiac myosin is evaluated by theeffect of the compound on the actin stimulated ATPase of S1 subfragment.Actin filaments in the assay are decorated with troponin and tropomyosinand Ca⁺⁺ concentration is adjusted to a value that would result in 50%of maximal activation. S1 ATPase is measured in the presence of adilution series of the compound. Compound concentration required for 40%activation above the ATPase rate measured in the presence of control(equivalent volume of DMSO) is reported as AC₄₀.

Example 11

In vivo Fractional Shortening Assay

Animals Male Sprague Dawley rats from Charles River Laboratories(275-350 g) are used for bolus efficacy and infusion studies. Heartfailure animals are described below. They are housed two per cage andhave access to food and water ad libitum. There is a minimum three-dayacclimation period prior to experiments.

Echocardiography Animals are anesthetized with isoflurane and maintainedwithin a surgical plane throughout the procedure. Core body temperatureis maintained at 37° C. by using a heating pad. Once anesthetized,animals are shaven and hair remover is applied to remove all traces offur from the chest area. The chest area is further prepped with 70% ETOHand ultrasound gel is applied. Using a GE System Vingmed ultrasoundsystem (General Electric Medical Systems), a 10 MHz probe is placed onthe chest wall and images are acquired in the short axis view at thelevel of the papillary muscles. 2-D M-mode images of the left ventricleare taken prior to, and after, compound bolus injection or infusion. Invivo fractional shortening ((end diastolic diameter−end systolicdiameter)/end diastolic diameter×100) is determined by analysis of theM-mode images using the GE EchoPak software program.

Bolus and infusion efficacy For bolus and infusion protocols, fractionalshortening is determined using echocardiography as described above. Forbolus and infusion protocols, five pre-dose M-Mode images are taken at30 second intervals prior to bolus injection or infusion of compounds.After injection, M-mode images are taken at 1 min and at five minuteintervals thereafter up to 30 min. Bolus injection (0.5-5 mg/kg) orinfusion is via a tail vein catheter. Infusion parameters are determinedfrom pharmacokinetic profiles of the compounds. For infusion, animalsreceived a 1 minute loading dose immediately followed by a 29 minuteinfusion dose via a tail vein catheter. The loading dose is calculatedby determining the target concentration×the steady state volume ofdistribution. The maintenance dose concentration is determined by takingthe target concentration×the clearance. Compounds are formulated in 25%cavitron vehicle for bolus and infusion protocols. Blood samples aretaken to determine the plasma concentration of the compounds.

Example 12

Hemodynamics in Normal and Heart Failure Animals

Animals are anesthetized with isoflurane, maintained within a surgicalplane, and then shaven in preparation for catheterization. An incisionis made in the neck region and the right carotid artery cleared andisolated. A 2 French Millar Micro-tip Pressure Catheter (MillarInstruments, Houston, Tex.) is cannulated into the right carotid arteryand threaded past the aorta and into the left ventricle. End diastolicpressure readings, max±dp/dt, systolic pressures and heart rate aredetermined continuously while compound or vehicle is infused.Measurements are recorded and analyzed using a PowerLab and the Chart 4software program (ADInstruments, Mountain View, Calif.). Hemodynamicsmeasurements are performed at a select infusion concentration. Bloodsamples are taken to determine the plasma concentration of thecompounds.

Example 13

Left Coronary Artery Occlusion Model of Congestive Heart Failure

Animals Male Sprague-Dawley CD (220-225 g; Charles River) rats are usedin this experiment. Animals are allowed free access to water andcommercial rodent diet under standard laboratory conditions. Roomtemperature is maintained at 20-23° C. and room illumination is on a12/12-hour light/dark cycle. Animals are acclimatized to the laboratoryenvironment 5 to 7 days prior to the study. The animals are fastedovernight prior to surgery.

Occlusion Procedure Animals are anaesthetized with ketamine/xylazine (95mg/kg and 5 mg/kg) and intubated with a 14-16-gauge modified intravenouscatheter. Anesthesia level is checked by toe pinch. Core bodytemperature is maintained at 37° C. by using a heating blanket. Thesurgical area is clipped and scrubbed. The animal is placed in rightlateral recumbency and initially placed on a ventilator with a peakinspiratory pressure of 10-15 cm H₂O and respiratory rate 60-110breaths/min. 100% O₂ is delivered to the animals by the ventilator. Thesurgical site is scrubbed with surgical scrub and alcohol. An incisionis made over the rib cage at the 4^(th)-5^(th) intercostal space. Theunderlying muscles are dissected with care to avoid the lateral thoracicvein, to expose the intercostal muscles. The chest cavity is enteredthrough 4^(th)-5^(th) intercostal space, and the incision expanded toallow visualization of the heart. The pericardium is opened to exposethe heart. A 6-0 silk suture with a taper needle is passed around theleft coronary artery near its origin, which lies in contact with theleft margin of the pulmonary cone, at about 1 mm from the insertion ofthe left auricular appendage. The left coronary artery is occluded bytying the suture around the artery (“LCO”). Sham animals are treated thesame, except that the suture is not tied. The incision is closed inthree layers. The rat is ventilated until able to ventilate on its own.The rats are extubated and allowed to recover on a heating pad. Animalsreceive buprenorphine (0.01-0.05 mg/kg SQ) for post operative analgesia.Once awake, they are returned to their cage. Animals are monitored dailyfor signs of infection or distress. Infected or moribund animals areeuthanized. Animals are weighed once a week.

Efficacy analysis Approximately eight weeks after infarction surgery,rats are scanned for signs of myocardial infarction usingechocardiography. Only those animals with decreased fractionalshortening compared to sham rats are utilized further in efficacyexperiments. In all experiments, there are four groups, sham+vehicle,sham+compound, LCL+vehicle and LCL+compound. At 10-12 weeks post LCL,rats are infused at a select infusion concentration. As before, fivepre-dose M-Mode images are taken at 30 second intervals prior toinfusion of compounds and M-mode images are taken at 30 second intervalsup to 10 minutes and every minute or at five minute intervalsthereafter. Fractional shortening is determined from the M-mode images.Comparisons between the pre-dose fractional shortening and compoundtreatment are performed by ANOVA and a post-hoc Student—Newman—Keuls.Animals are allowed to recover and within 7-10 days, animals are againinfused with compounds using the hemodynamic protocol to determinehemodynamic changes of the compounds in heart failure animals. At theend to the infusion, rats are killed and the heart weights determined.

When tested as described above, compounds of Formula I are shown to havethe desired activity.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto. All patents and publications cited above arehereby incorporated by reference.

1. A compound represented by Formula I:

wherein: R¹, R² and the nitrogen to which they are attached form apiperidine-1-yl ring, wherein the piperidin-1-yl ring is optionallysubstituted with one, two or three of the following groups: alkyl,halogen, hydroxy, alkoxy, alkylenedioxy, carboxy, acyloxy,alkoxycarbonyl, alkoxycarbonylamino, aminocarbonyl, cyano, acyl, oxo,nitro, amino, sulfanyl, sulfinyl, sulfonyl, aminosulfonyl, amidino,phenyl, benzyl, heteroaryl, heterocyclyl, aryloxy, arallkoxy,heteroaryloxy, and heteroaralkoxy; R³ is an aryl or heteroaryl group,which is optionally substituted with a halogen, lower alkoxy, aryl orheteroaryl group; R⁴ is halogen; R⁵ is hydrogen, halogen, hydroxy, orlower alkyl; and R⁶ and R⁷ are independently selected from hydrogen,halogen, hydroxy, and lower alkyl; or a pharmaceutically acceptable saltthereof.
 2. The compound of claim 1, or a pharmaceutically acceptablesalt thereof, wherein R³ is a phenyl, isoxazolyl, oxazolyl, pyridinyl,pyrazinyl, pyrimidinyl, tetrazol-5-yl, thiazolyl, thiadiazolyl orimidazolyl group, which is optionally substituted with a halogen, loweralkoxy, aryl or heteroaryl group.
 3. The compound of claim 1, or apharmaceutically acceptable salt thereof, wherein R³ is an[1,3,4]thiadiazol-2-yl group which is optionally substituted with aphenyl group.
 4. The compound of claim 1, or a pharmaceuticallyacceptable salt thereof, wherein R³ is 5-phenyl-[1,3,4]thiadiazol-2-yl.5. The compound of claim 4, or a pharmaceutically acceptable saltthereof, wherein R⁴ is chloro; and R⁵, R⁶ and R⁷ are hydrogen.
 6. Thecompound of claim 1, or a pharmaceutically acceptable salt thereof,wherein R³ is a 1H-imidazol-2-yl group.
 7. The compound of claim 6, or apharmaceutically acceptable salt thereof, wherein R⁴ is chloro; and R⁵,R⁶ and R⁷ are hydrogen.
 8. The compound of claim 1, or apharmaceutically acceptable salt thereof, wherein R³ is oxazol-2-yl. 9.The compound of claim 8, or a pharmaceutically acceptable salt thereof,wherein R⁴ is chloro; and R⁵, R⁶ and R⁷ are hydrogen.
 10. The compoundof claim 1, or a pharmaceutically acceptable salt thereof, wherein R⁴ ischloro; and R⁵, R⁶ and R⁷ are hydrogen.
 11. A pharmaceutical formulationcomprising a pharmaceutically accepted excipient and a therapeuticallyeffective amount of a compound of claim 1, or a pharmaceuticallyacceptable salt thereof.